Self-regulating aav vectors for safe expression of mecp2 in rett syndrome

ABSTRACT

In some aspects, the disclosure relates to compositions and methods of engineering a transgene. In some embodiments, the disclosure provides self-regulating recombinant nucleic acids, viral vectors and pharmaceutical compositions comprising a MeCP2 transgene. In some embodiments, compositions and methods described by the disclosure are useful for treating diseases and disorders associated with a loss of function mutation, for example Rett syndrome.

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(c) of thefiling date of U.S. Provisional Application Ser. No. 62/516,060, filedon Jun. 6, 2017, the entire contents of which are incorporated herein byreference.

BACKGROUND

Rett syndrome is a neurological disease caused by loss of functionmutations in MeCP2. It has been observed that post-natal restoration ofMeCP2 expression is effective in reversing some of the phenotypespresent in MeCP2 mice, but safety concerns remain. Additionally, studiesexamining the therapeutic efficacy of certain MeCP2 vectors encoding thee1isoform have observed only partial rescue of Rett phenotypes. Thesestudies have typically focused on neonatal intravascular (IV) orintracerebroventricular (ICV) delivery, and in some instances haveencountered lethal liver toxicity and hindlimb clasping.

SUMMARY

Aspects of the disclosure relate to the discovery that certaincombinations of miRNA regulatory elements (MREs), for example miRNAbinding sites associated with gene expression negative feedback loopsand miRNA binding sites that de-target transgene expression fromnon-target tissues, enable tunable transgene expression within a narrowrange compatible with normal protein function and avoidance ofoff-target transgene toxicity. In some embodiments, compositions andmethods described by the disclosure are therefore useful for treatingdiseases and disorders associated with loss of function mutations, forexample Rett syndrome which is associated with loss of functionmutations in the MECP2 gene.

Accordingly, in some aspects, the disclosure provides a method ofengineering a transgene, the method comprising: selecting a first geneencoding a first product in a cell selecting a second gene encoding asecond product in the cell; determining that expression of the secondproduct is positively regulated by the first product in the cell;selecting an miRNA; determining that expression of the miRNA ispositively regulated by the second product in the cell; and, engineeringa transgene to express in the cell a transcript having a coding regionencoding the first product and having one or more binding sites for themiRNA.

In some aspects, the disclosure provides a method of engineering atransgene, the method comprising: selecting a first gene encoding afirst product in a cell; selecting an miRNA, the expression of which ispositively regulated by the first product in the cell; and, engineeringa transgene that expresses a transcript having a coding region encodingthe first product and a 3′-non-coding region comprising one or morebinding sites for the miRNA.

In some aspects, the disclosure provides a method of engineering atransgene, the method comprising: selecting a first gene encoding afirst product in a cell; selecting a second gene encoding a secondproduct in the cell; determining that expression of the second productis positively regulated by the first product in the cell; selecting anmiRNA; determining that expression of the miRNA is positively regulatedby the second product in the cell; and, engineering a transgene toexpress in the cell a transcript having a coding region encoding thefirst product and a 3′-non-coding region comprising one or more bindingsites for the miRNA.

In some aspects, the disclosure provides a recombinant nucleic acidencoding a transcript having i) a coding region encoding a protein andii) two or more miRNA binding sites, wherein the two or more miRNAbinding sites comprise: at least one first, miRNA binding site specificfor a first miRNA that is positively regulated by expression of theprotein in a cell of a target tissue; and at least one second miRNAbinding site specific for a second miRNA that is expressed, independentof expression of the protein, in cells of a non-target tissue.

In some aspects, the disclosure provides a recombinant nucleic acidencoding a transcript having a coding region encoding human MeCP2protein or a functional fragment thereof and a 3′-non-coding regioncomprising one or more miRNA binding sites, wherein the one or moremiRNA binding sites comprise: at least one miRNA binding site specificfor an miRNA that negatively regulates expression of the transcript; andat least one miRNA binding site specific for an miRNA that inhibitsexpression of the transcript in a non-target tissue.

In some aspects, the disclosure provides a recombinant nucleic acidencoding a transcript having: a coding region encoding human MeCP2 or afunctional fragment thereof and, a 3′-non-coding region comprising oneor more miRNA binding sites, wherein transcript is flanked byadeno-associated virus (AAV) inverted terminal repeats (ITRs). In someembodiments, an AAV ITR is an AAV2, AAV3, AAV4, AAV5, or AAV6 ITR. Insome embodiments, AAV ITRs are AAV2 ITRs. In some embodiments, ITRs areartificial sequences that replace ITR function, for example as disclosedin WO/2016/17208).

In some aspects, the disclosure provides a viral vector comprising arecombinant nucleic acid as described by the disclosure. In someembodiments, a viral vector is an adeno-associated virus (AAV) vector,an adenovirus vector, a lentiviral vector, a herpesvirus vector, or abaculovirus vector.

In some aspects, the disclosure provides a recombinant adeno-associatedvirus (rAAV) comprising: a recombinant nucleic acid as described by thedisclosure; at least one adeno-associated virus (AAV) inverted terminalrepeat (ITR); and a capsid protein.

In some aspects, the disclosure provides a recombinant AAV (rAAV) vectorfor self-regulated expression of a protein, the rAAV vector comprising anucleic acid engineered to express in a cell of a target tissue atranscript encoding the protein, wherein the transcript comprises atleast one first miRNA binding site specific for a first miRNA, whereinexpression of the first miRNA is positively regulated by expression ofthe protein in the cell.

In some aspects, the disclosure provides a composition comprising arecombinant nucleic acid as described by the disclosure, or an rAAV asdescribed by the disclosure, and a pharmaceutically acceptableexcipient. In some embodiments, a composition is formulated forinjection, for example systemic injection (e.g., intravenous injection)or intrathecal injection.

In some embodiments, a first product is a protein. In some embodiments,the protein is MeCP2, for example MeCP2 isoform e1 or MeCP2 isoform e2.In some embodiments, a first product is an miRNA or a long non-codingRNA.

In some embodiments, a second product is a protein, or nucleic acid. Insome embodiments, the second product is bone-derived neurotrophic factor(BDNF). In some embodiments, the nucleic acid is an miRNA (e.g.,miR-132). In some embodiments, the first miRNA is miR-132.

In some embodiments, at least one miRNA binding site specific for anmiRNA that negatively regulates expression of the transcript comprises amiR-132 binding site, for example two or three miR-132 binding sites.

In some embodiments, at least one miRNA binding site specific for anmiRNA that inhibits expression of the transcript in a non-target tissuecomprises a miR-1 binding site, mir-122 binding site, or miR-1 andmiR-122 binding site. In some embodiments, the at least one miRNAbinding site specific for an miRNA that inhibits expression of thetranscript in a non-target tissue comprises three miR-1 binding sites(e.g., 3×-miR-1) and three niR-122 binding sites (e.g., 3×-miR-122).

In some embodiments, methods described by the disclosure furthercomprise the step of engineering the 3′-non-coding region of thetranscript to comprise one or more binding sites for one or morede-targeting miRNAs. In some embodiments, one or more de-targetingmiRNAs inhibit expression of the transgene from liver, heart, lung,muscle, pancreas, or immune (e.g., antigen presenting) cells. In someembodiments, one or more de-targeting miRNA is miR-122, miR-1, ormiR-122 and miR-1. In some embodiments, one or more de-targeting miRNAsinhibit expression of the transgene in immune cells, such as antigenpresenting cells (e.g., dendritic cells, macrophages, etc.). In someembodiments, one or more de-targeting miRNA is miR-15a, miR-16-1,miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1, miR-21, miR-29a, miR-29b,miR-29c, miR-30b, miR-31, miR-34a, miR-92a-1, miR-106a, miR-125a,miR-125b, miR-126, miR-142-3p, miR-146a, miR-150, miR-155, miR-181a,miR-223 or miR-424.

In some embodiments, an miRNA binding site or miRNA binding sites islocated between the last codon of the coding region and the poly-A tailof the transcript.

In some embodiments of methods described by the disclosure, the step ofengineering the transgene comprises inserting the transgene into avector. In some embodiments, a vector is a cloning vector, expressionvector, plasmid, or viral vector.

In some embodiments, a recombinant nucleic acid further comprises apromoter, for example a mouse MeCP2 promoter. In some embodiments, amouse MeCP2 promoter comprises the sequence set forth in SEQ ID NO: 3.

In some embodiments, a recombinant nucleic acid is located on a plasmid.

In some embodiments, a capsid protein is a capsid protein thatfacilitates crossing of the rAAV across the blood-brain barrier of asubject. In some embodiments, a capsid protein has a serotype selectedfrom the group consisting of AAV-PHP.B. AAV1, AAV2. AAV2i8, AAV2.5,AAV5, AAV6, AAV8. AAVrh8. AAV9. AAVrh10, AAV-B1, AAV9.45A-String (e.g.,AAV9.45-AS), AAV9.45Angiopep, AAV9.47-Angiopep. and AAV9.47-AS. AAV5,AAVrh39. AAVrh43, CAM130, and AAV9HR. In some embodiments, a capsidprotein has a serotype as described in WO2015/127128. WO2016/054554,WO2016054557, or WO2016/065001. In some embodiments, a capsid proteincomprises or consists of a sequence set forth in SEQ ID NO: 14 or 15(e.g., AAV-PHP.B or AAV9).

In some aspects, the disclosure provides a method of treating Rettsyndrome in a subject, the method comprising, administering to a subjecthaving or suspected of having Rett syndrome an effective amount of: arecombinant nucleic acid as described by the disclosure; a rAAV asdescribed by the disclosure; or, a composition as described by thedisclosure.

In some embodiments, the subject is a human subject. In someembodiments, a subject is less than one year old. In some embodiments, asubject is characterized by a mutation in at least one copy of the MeCP2gene, for example a loss of function mutation.

In some embodiments, a recombinant nucleic acid, rAAV or composition asdescribed by the disclosure is administered by injection, for examplesystemic injection (e.g., intravenous injection) or intrathecalinjection. In some embodiments, the administration results in theeffective amount of the recombinant nucleic acid, rAAV or compositioncrossing the blood-brain barrier of a subject. In some embodiments, theadministration results in a non-toxic level of MeCP2 expression in thebrain of the subject.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-1B show characterization of new AAV-MeCP2 vectors for safe andeffective gene therapy in Rett syndrome. FIG. 1A shows a schematicdepiction of a homeostatic mechanism of MeCP2 auto-regulation. FIG. 1Bshows a schematic depiction of the structure of self-regulated AAV-MeCP2vectors encoding human MeCP2-e1 with a myc tag under a mouse MeCP2promoter (−223 to +56) and different microRNA recognition elements(e.g., miR-122/IT; miR-132T).

FIGS. 2A-2C show effective expression of AAV2-MeCP2 in HEK293T cellsFIG. 2A shows MeCP2 expression measured by Western blot. FIG. 2B showsMeCP2 expression measured by a normalized protein expression assay (FIG.211 ). FIG. 2C shows a toxicity profile of 293T cells transduced withAAV2-MeCP2 for four days at a dose of 30.00) gc/cell.

FIGS. 3A-3C show AAV2-MeCP2 expression in mouse cortical neurons. FIG.3A shows mouse primary cortical neurons were transduced at AAV vectordoses ranging from 1E3-1E5 vg/cell including AAV-GFP as a control. FIG.3B shows Western blot analysis of hMeCP2-myc expression in neurons 5days after infection with 3E4 vector genomes(dose)/cell. FIG. 3C showsmiR-132 expression in response to AAV2-MeCP2 re-delivery.

FIGS. 4A-4C show representative data obtained from in vivo mouseexperiments. FIG. 4A shows wild-type post-natal day 1 mice injected viathe facial vein with AAV encoding the e1 isoform of human MeCP2containing 0, 1×, 2×, or 3× miR-132 target sequences. Wild-type animalswere euthanized 3 months following injection and whole brain, heart andliver tissue was subjected to total RNA extraction, cDNA synthesis andqRT-PCR using primers specific to the e1 isoform of human MeCP2. Datawere normalized to AAV-MeCP2 containing 3×miR-132 target sequences,which was set to 1. FIG. 4B shows gene expression analysis of humanMeCP2 isoform e1 in brain of wild-type mice following intracranialinjection of AAV-MeCP2.

FIG. 4C shows gene expression analysis of human MeCP2 isoform e1 inbrain of wild-type mice following intracranial injection of AAV-MeCP2.

FIG. 5 shows MeCP2 expression driven by constructs described by thedisclosure is effectively de-targeted from heart and liver.

DETAILED DESCRIPTION

Aspects of the disclosure relate, in part, to AAV vectors capable ofself-regulating transgene (e.g., MeCP2) expression levels to preventoverexpression related toxicity. In some embodiments, theself-regulating mechanism is based on the presence of multiple copies ofa miRNA regulatory element (e.g., one or more miR-132 binding sites) inthe 3′UTR of the transgene cassette. As described further in theExamples section. AAV vectors capable of self-regulating transgeneexpression, in some embodiments, have an improved efficacy and safetyprofile compared to other AAV vectors, for example AAV vectorscomprising native transgene promoters only. It should be recognized thatthe observations described in the Examples section in the context ofmiR-132/MeCP2 constructs is applicable to other transgene expressionconstructs comprising binding sites of other miRs that regulate proteinexpression (e.g., through a negative feedback loop).

In some embodiments, delivery routes that are most likely to mediateglobal gene delivery to the CNS (e.g., systemic injection andintrathecal injection) are likely to result in high level transductionof peripheral organs where transgene (e.g., MeCP2) expression may becometoxic. The disclosure is based, in part, on the recognition thatcombining miRNA regulatory elements (MREs), such as miRNA binding sites(e.g., miR-122 binding sites and miR-1 binding sites), with MREsassociated with negative feedback loops regulating protein expression(e.g., miR-132 binding sites for MeCP2), simultaneously regulatetransgene expression levels and de-target transgene expression inperipheral organs.

Accordingly in some aspects, the disclosure provides a method ofengineering a transgene, the method comprising: selecting a first geneencoding a first product in a cell; selecting an miRNA, the expressionof which is positively regulated by the first product in the cell; and,engineering a transgene that expresses a transcript having a codingregion encoding the first product and one or more binding sites for themiRNA. In some embodiments, the one or more binding sites for the miRNAare located in a 3′-non-coding region of the transcript.

In some aspects, the disclosure provides a method of engineering atransgene, the method comprising: selecting a first gene encoding afirst product in u cell; selecting a second gene encoding a secondproduct in the cell; determining that expression of the second productis positively regulated by the first product in the cell; selecting anmiRNA; determining that expression of the miRNA is positively regulatedby the second product in the cell; and, engineering a transgene toexpress in the cell a transcript having a coding region encoding thefirst product and one or more binding sites for the miRNA. In someembodiments, the one or more binding sites for the miRNA are located ina 3′-non-coding region of the transcript.

As used herein, “engineering a transgene” refers to production (e.g.,synthesis) of a recombinant nucleic acid using gene cloning techniques,such as polymerase chain reaction (PCR) restriction enzyme digestion,and in vitro nucleic acid ligation, for example as described in Sambrooket al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press.Cold Spring Harbor. N.Y.

As used herein, a “product” or “gene product” refers to a nucleic acid(e.g., RNA transcript, dsRNA, miRNA, etc.), a peptide, protein, orpolypeptide that is transcribed and/or translated from a nucleic acid(e.g., DNA or RNA) sequence. In some embodiments, a product is an RNAtranscript comprising a protein coding region. In some embodiments, aprotein coding region encodes a protein associated with a disease causedby a loss of function mutation (e.g., MeCP2). Additional examples ofproteins associated with a disease caused by a loss of function mutationinclude but are not limited to tyrosinase (Tyrosinemia), lysosomal acidbeta-galactosidase (GM1-gangliosidosis) beta-hexosaminidase A and B(Tay-Sach and Sandhoff disease), aspartoacylase (ASPA; Canavan disease).Aspartylglucosamininidase (Aspartylglucosaminuria), Palmitoyl proteinthioesterase (Infantile Batten disease), tripeptidyl peptidase (Lateinfantile Batten disease), α-Galactosidase (Fabry disease), α-Fucosidase(Fucosidosis). Protective protein/cathepsin A (Galactosialidosis),β-Glucosidase (Gaucher disease). Galactosylceramidase (Globoid-cellleukodystrophy), α-Mannosidase (α-Mannosidosis), Arylsulfatase A(Metachromatic leukodystrophy), α-L-iduronidase (MucopolysaccharidosisI). α-N-acetylglucosaminidase (Mucopolysaccharidosis IIIB).Arylsulfatase B (Mucopolysaccharidosis VI). β-Glucuronidase(Mucopolysaccharidosis VID. Acid sphingomyelinase (Nieman-Pick disease),α-Glucosidase (Pompe disease) and Acid lipase (Wolman disease), FOXG1(FOXG1 Syndrome), CDKL5, N-GlyI, Glut-1 (De Vivo disease), etc.

In some embodiments, a product is an interfering nucleic acid, forexample a miRNA that regulates expression or activity of a gene product.

In some embodiments, one product regulates gene expression or proteinexpression of a second product. Regulation of gene product expression ortranslation can be positive or negative. “Positive regulation” refers toan increase of gene expression or activity (e.g., as a result of theexpression or activity of another gene product). “Negative regulation”refers to a decrease or inhibition of gene expression or activity (e.g.,as a result of the expression or activity of another gene productthrough a feedback loop).

In some embodiments, gene products such as growth factors, transcriptionfactors (e.g., as described in Wang et al. Nucleic Acids Res. 2010January; 38 (Database issue): D119-D122), etc. are capable of regulatingtransgene expression or activity in a cell or subject. Examples ofgrowth factors include neurotrophins, such as brain-derived neurotrophicfactor (BDNF), nerve growth factor (NGF), neurotrophin 3, neurotrophin4, glial derived neurotrophic factor (GDNF), ciliary neurotrophic factor(CNTF), fibroblast growth factors (FGF1 to 23), neurturin, insulin-likegrowth factor 1 (IGF-1), insulin-like growth factor 2 (IGF-2), etc.

In some embodiments, transgenes as described by the disclosure areengineered to comprise at least one miRNA regulatory element (e.g.,miRNA binding site) that is associated with a gene expression regulatoryloop (e.g., negative feedback loops, positive feedback loops, etc.).Generally, gene expression regulatory loops may be endogenous to a cell,or artificial (e.g., one or more elements of the feedback loop areprovided along with a transgene). In one example of a negative feedbackloop, expression of MeCP2 in a cell causes an increase of brain-derivedneurotrophic factor (BDNF) in the cell, which in turn increasesexpression of miR-132, which in turn regulates MeCP2 expression (FIG. 1). It should be appreciated that, in some embodiments, the disclosurerelates to positive feedback loops, which may be used to amplifytransgene expression.

In some embodiments, transgenes as described by the disclosure areengineered to comprise at least one miRNA regulatory element (e.g.,miRNA binding site) that de-targets expression of the transgene from oneor more non-target tissues. As used herein, “non-target tissue” refersto a tissue (e.g., cells of a tissue) in which expression of thetransgene is undesirable. For example, in some embodiments,overexpression of MeCP2 in a cell results in hepatic cytotoxicity; inthat context, liver tissue (e.g., liver cells) are a non-target tissue.In some embodiments, a non-target tissue is liver (e.g., liver cells),heart (e.g., heart cells), pancreas (e.g., pancreatic cells), muscle(e.g., muscle cell), immune cell (e.g., antigen presenting cells, etc.),or any combination thereof.

As used herein, “target tissue” refers to a tissue (e.g., cells of atissue) in which expression of a transgene is preferred relative toother tissues, such as non-target tissues. In some embodiments, a targettissue is CNS tissue (e.g., CNS cells, such as neurons). Non-limitingexamples of CNS tissue include brain tissue (e.g., neurons, glial cells,etc.) and spinal cord tissue.

Generally, the one or more miRNA binding sites of a transcript encodedby a transgene are located in the 3′ untranslated region (3′UTR) of thetranscript. In some embodiments, the one or more miRNA binding sites arelocated between the last codon of the coding region of the transcriptand the poly-A tail of the transcript. However, it should be appreciatedthat, in some embodiments, one or more miRNA binding sites are locatedin a region other than the 3′UTR of the transcript, for example in anintron at the 5′-end of the transcript. The number of miRNA bindingsites engineered into a transgene as described by the disclosure willvary depending upon the gene product encoded by the transgene, and maybe determined empirically by a skilled artisan without an undue amountof experimentation. For example, in some embodiments a transgene asdescribed by the disclosure comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10miRNA binding sites. In some embodiments a transgene as described by thedisclosure comprises more than 10 (e.g., any integer between 11 and 100)miRNA binding sites. In some embodiments, a transgene as described bythe disclosure comprises 3, 4, or 5 miRNA binding sites. The one or moremiRNA binding sites may each bind the same miRNA, or different miRNA. Insome embodiments, a transgene as described by the disclosure comprisesone or more (e.g. 3) miR-122 binding site(s), one or more (e.g., 3)miR-1 binding site(s) and three miR-132 binding sites.

Recombinant Nucleic Acids

In some embodiments, a transgene as described by the disclosure isencoded by a recombinant nucleic acid. A “nucleic acid” sequence refersto a DNA or RNA sequence. In some embodiments, proteins and nucleicacids of the disclosure are isolated. As used hemin, the term “isolated”means artificially produced. As used herein with respect to nucleicacids, the term “isolated” means: (i) amplified in vitro by, forexample, polymerase chain reaction (PCR); (ii) recombinantly produced bycloning; (iii) purified, as by cleavage and gel separation; or (iv)synthesized by, for example, chemical synthesis. An isolated nucleicacid is one which is readily manipulable by recombinant DNA techniqueswell known in the art. Thus, a nucleotide sequence contained in a vectorin which 5′ and 3′ restriction sites are known or for which polymerasechain reaction (PCR) primer sequences have been disclosed is consideredisolated but a nucleic acid sequence existing in its native state in itsnatural host is not. An isolated nucleic acid may be substantiallypurified, but need not be. For example, a nucleic acid that is isolatedwithin a cloning or expression vector is not pure in that it maycomprise only a tiny percentage of the material in the cell in which itresides. Such a nucleic acid is isolated, however, as the term is usedherein because it is readily manipulable by standard techniques known tothose of ordinary skill in the art. As used herein with respect toproteins or peptides, the term “isolated” refers to a protein or peptidethat has been isolated from its natural environment or artificiallyproduced (e.g., by chemical synthesis, by recombinant DNA technology,etc.).

The skilled artisan will also realize that conservative amino acidsubstitutions may be made to provide functionally equivalent variants,or homologs of the capsid proteins. In some aspects the disclosureembraces sequence alterations that result in conservative amino acidsubstitutions. As used herein, a conservative amino acid substitutionrefers to an amino acid substitution that does not alter the relativecharge or size characteristics of the protein in which the amino acidsubstitution is made. Variants can be prepared according to methods foraltering polypeptide sequence known to one of ordinary skill in the artsuch as are found in references that compile such methods, e.g.,Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds.,Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,New York, 1989, or Current Protocols in Molecular Biology, F. M.Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservativesubstitutions of amino acids include substitutions made among aminoacids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K,R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. Therefore, one canmake conservative amino acid substitutions to the amino acid sequence ofthe proteins and polypeptides disclosed herein.

In some embodiments, a nucleic acid as described by the disclosure iscontained within a vector. As used herein, the term “vector” includesany genetic element, such as a plasmid, phage, transposon, cosmid,chromosome, artificial chromosome, virus, virion, etc., which is capableof replication when associated with the proper control elements andwhich can transfer gene sequences between cells. Thus, the term includescloning and expression vehicles, as well as viral vectors. Examples ofviral vector include adenovirus vector, adeno-associated virus (AAV)vector, lentiviral vectors, herpesvirus vectors, baculovirus vectors,etc.

MeCP2

In some aspects, the disclosure relates to compositions and methods forexpressing MeCP2 protein in a cell or subject. “MeCP2” refers to methylCpG binding protein 2, which is encoded by the MeCP2 gene and playsimportant roles (e.g., functions as a transcriptional repressor, ortranscriptional activator) in nerve cells, such as mature neurons. Oneexample of a MeCP2 gene is represented by GenBank Accession NumberNM_001110792 (MeCP2-e1). Another example of a MeCP2 gene is representedby GenBank Accession Number NM_001110792 (MeCP2-e2). The MeCP2 geneencodes two isoforms of MeCP2 protein, referred to as MeCP2 isoform e1and MeCP2 isoform e2, which differ in the length of their N-terminus. Insome embodiments, MeCP2 isoform e1 is represented by a sequence setforth in SEQ ID NO: 1. In some embodiments, MeCP2 isoform c2 isrepresented by a sequence set forth in SEQ ID NO: 2.

In some embodiments, a transgene (e.g., a recombinant nucleic acid)encodes a functional fragment of MeCP2 protein (e.g., a fragment ofisoform e1 or isoform e2). A “functional fragment” of MeCP2 is atruncated MeCP2 protein that retains the natural function (e.g.,transcriptional activator or transcriptional repressor) of wild-type(e.g., full-length) MeCP2 protein. In some embodiments, a functionalfragment of MeCP2 comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more aminoacid truncations relative to full-length MeCP2 protein. In someembodiments, a functional fragment of MeCP2 comprises between about 1and 10, 5 and 50, 20 and 100 amino acid truncations relative tofull-length MeCP2 protein.

In some embodiments, a transgene (e.g., a recombinant nucleic acid)encodes a variant of MeCP2 protein (e.g., a variant of isoform e1 orisoform e2). A variant of MeCP2 protein may have between about 50% andabout 99.9% identity to a wild-type MeCP2 protein (e.g., SEQ ID NO: 1 orSEQ 1D NO: 2). In some embodiments, a MeCP2 variant has about 50%, about60%, about 70%, about 80%, about 90%, about 95%, or about 99% identitywith a wild-type MeCP2 protein (e.g., SEQ ID NO: 1 or SEQ 1D NO: 2).

miRNA and miRNA Binding Sites

The disclosure is based, in part, on the recognition that combiningmiRNA regulatory elements (MREs), such as miRNA binding sites (e.g.,miR-122 binding sites and miR-1 binding sites), with MREs associatedwith negative feedback loops regulating protein expression (e.g.,miR-132 binding sites for MeCP2), simultaneously regulate transgeneexpression levels and de-target transgene expression in peripheralorgans.

miRNAs and other small interfering nucleic acids regulate geneexpression via target RNA transcript cleavage/degradation ortranslational repression of the target messenger RNA (mRNA), miRNAs arenatively expressed, typically as final 19-25 non-translated RNAproducts, miRNAs exhibit their activity through sequence-specificinteractions with the 3′ untranslated regions (UTR) of target mRNAs.These endogenously expressed miRNAs form hairpin precursors which aresubsequently processed into a miRNA duplex, and further into a “mature”single stranded miRNA molecule. This mature miRNA guides a multiproteincomplex, miRISC, which identifies target site, e.g., in the 3′ UTRregions, of target mRNAs based upon their complementarity to the maturemiRNA.

The following non-limiting list of miRNA genes, and their homologues,are useful in methods and compositions of the disclosure (e.g., formediating self-regulated expression or de-targeting of a transgene):hsa-let-7a, hsa-let-7a*, hsa-let-7b, hsa-let-7b*, hsa-let-7c,hsa-let-7c*, hsa-let-7d, hsa-let-7d*, hsa-let-7e, hsa-let-7e,hsa-let-7f, hsa-let-7f-1*, hsa-let-7f-2*, hsa-let-7g, hsa-let-7g*,hsa-let-7i, hsa-let-7i*, hsa-miR-1, hsa-miR-100, hsa-miR-100*,hsa-miR-101, hsa-miR-101*, hsa-miR-103, hsa-miR-105, hsa-miR-105*,hsa-miR-106a, hsa-miR-106a*, hsa-miR-106b, hsa-miR-106b*, hsa-miR-107,hsa-miR-10a, hsa-miR-10a*, hsa-miR-10b, hsa-miR-10b*, hsa-miR-1178,hsa-miR-1179, hsa-miR-1180, hsa-miR-1181, hsa-miR-1182, hsa-miR-1183,hsa-miR-1184, hsa-miR-1185, hsa-miR-1197, hsa-miR-1200, hsa-miR-1201,hsa-miR-1202, hsa-miR-1203, hsa-miR-1204, hsa-miR-1205, hsa-miR-1206,hsa-miR-1207-3p, hsa-miR-1207-5p, hsa-miR-1208, hsa-miR-122,hsa-miR-122*, hsa-miR-1224,3p, hsa-miR-1224-5p, hsa-miR-1225-3p,hsa-miR-1225-5p, hsa-miR-1226, hsa-miR-1226*, hsa-miR-1227,hsa-miR-1228, hsa-miR-1228*, hsa-miR-1229, hsa-miR-1231, hsa-miR-1233,hsa-miR-1234, hsa-miR-1236, hsa-miR-1237, hsa-miR-1238, hsa-miR-124,hsa-miR-124*, hsa-miR-1243, hsa-miR-1244, hsa-miR-1245, hsa-miR-1246,hsa-miR-1247, hsa-miR-1248, hsa-miR-1249, hsa-miR-1250, hsa-miR-1251,hsa-miR-1252, hsa-miR-1253, hsa-miR-1254, hsa-miR-1255a, hsa-miR-1255b,hsa-miR-1256, hsa-miR-1257, hsa-miR-1258, hsa-miR-1259, hsa-miR-125a-3p,hsa-miR-125a-5p, h % a-miR-125b, hsa-miR-125b-1*, hsa-miR-125b-2*,hsa-miR-126, hsa-miR-126*, hsa-miR-1260, hsa-miR-1261, hsa-miR-1262,hsa-miR-1263, hsa-miR-1264, hsa-miR-1265, hsa-miR-1266, hsa-miR-1267,hsa-miR-1268, hsa-miR-1269, hsa-miR-1270, hsa-miR-1271, hsa-miR-1272,hsa-miR-1273, hsa-miR-127-3p, hsa-miR-1274a, hsa-miR-1274b,hsa-miR-1275, hsa-miR-127-5p, hsa-miR-1276, hsa-miR-1277, hsa-miR-1278,hsa-miR-1279, hsa-miR-128, hsa-miR-1280, hsa-miR-1281, hsa-miR-1282,hsa-miR-1283, hsa-miR-1284, hsa-miR-1285, hsa-miR-1286, hsa-miR-1287,hsa-miR-1288, hsa-miR-1289, hsa-miR-129*, hsa-miR-1290, hsa-miR-1291,hsa-miR-1292, hsa-miR-1293, hsa-miR-129-3p, hsa-miR-1294, hsa-miR-1295,hsa-miR-129-5p, hsa-miR-1296, hsa-miR-1297, hsa-miR-1298, hsa-miR-1299,hsa-miR-1300, hsa-miR-1301, hsa-miR-1302, hsa-miR-1303, hsa-miR-1304,hsa-miR-1305, hsa-mi R-1306, hsa-miR-1307, hsa-miR-1308, hsa-miR-130a,hsa-miR-130a*, hsa-miR-130b, hsa-miR-130b*, hsa-miR-132, hsa-miR-132*,hsa-miR-1321, hsa-miR-1322, hsa-miR-1323, hsa-miR-1324, hsa-miR-133a,hsa-miR-133b, hsa-miR-134, hsa-miR-135a, hsa-miR-135a*, hsa-miR-135b,hsa-miR-135b*, hsa-miR-136, hsa-miR-136*, hsa-miR-137, hsa-miR-138,hsa-miR-138-1*, hsa-miR-138-2*, hsa-miR-139-3p, hsa-miR-139-5p,hsa-miR-140-3p, hsa-miR-140-5p, hsa-miR-141, hsa-miR-141*,hsa-miR-142-3p, hsa-miR-142-5p, hsa-miR-143, hsa-miR-143*, hsa-miR-144,hsa-miR-144*, hsa-miR-145, hsa-miR-145*, hsa-miR-146a, hsa-miR-146a*,hsa-miR-146b-3p, hsa-miR-146b-5p, hsa-miR-147, hsa-miR-147b,hsa-miR-148a, hsa-miR-148a*, hsa-miR-148b, hsa-miR-148b*, hsa-miR-149,hsa-miR-149*, hsa-miR-150, hsa-miR-150*, hsa-miR-151-3p, hsa-MiR-151-5p,hsa-miR-152, hsa-miR-153, hsa-miR-154, hsa-miR-154*, hsa-miR-155,hsa-miR-155*, hsa-miR-1Sa, hsa-miR-15a*, hsa-miR-15b, hsa-miR-15b*,hsa-miR-16, hsa-miR-16-1*, hsa-miR-16-2*, hsa-miR-17, hsa-miR-17*,hsa-miR-181a, hsa-miR-181a*, hsa-miR-181a-2*, hsa-miR-181b,hsa-miR-181c, hsa-miR-181c*, hsa-miR-181d, hsa-miR-182, hsa-miR-182*,hsa-miR-1825, hsa-miR-1826, hsa-miR-1827, hsa-miR-183, hsa-miR-183*,hsa-miR-184, hsa-miR-185, hsa-miR-185*, hsa-miR-186, hsa-miR-186*,hsa-miR-187, hsa-miR-187*, hsa-miR-188-3p, hsa-miR-188-5p, hsa-miR-18a,hsa-miR-18a*, hsa-miR-18b, hsa-miR-18b*, hsa-miR-190, hsa-miR-190b,hsa-miR-191, hsa-miR-191*, hsa-miR-192, hsa-miR-192*, hsa-miR-193a-3p,hsa-miR-193a-5p, hsa-miR-193b, hsa-miR-193b*, hsa-miR-194, hsa-miR-194*,hsa-miR-195, hsa-miR-195*, hsa-miR-196a, hsa-miR-1,96a*, hsa-miR-196b,hsa-miR-197, hsa-miR-198, hsa-miR-199a-3p, hsa-miR-199a-5p,hsa-miR-199b-5p, hsa-miR-19a, hsa-miR-19a*, hsa-miR-19b, hsa-miR-19b-1*,hsa-miR-19b-2*, hsa-miR-200a, hsa-miR-200a*, hsa-miR-200b,hsa-miR-200b*, hsa-miR-200k, hsa-miR-200c*, hsa-miR-202, hsa-miR-202*,hsa-miR-203, hsa-miR-204, hsa-miR-205, hsa-miR-206, hsa-miR-208a,hsa-miR-208b, hsa-miR-20a, hsa-miR-20a*, hsa-miR-20h, hsa-miR-20b*,hsa-miR-21, hsa-miR-21*, hsa-miR-210, hsa-miR-211, hsa-miR-212,hsa-miR-214, hsa-miR-214*, hsa-miR-215, hsa-miR-216a, hsa-miR-216b,hsa-miR-217, hsa-miR-218, hsa-miR-218-1*, hsa-miR-218-2*,hsa-miR-219-1-3p, hsa-miR-219-2-3p, hsa-miR-219-5p, hsa-miR-22,hsa-miR-22*, hsa-miR-220a, hsa-miR-220b, hsa-miR-220c, hsa-miR-221,hsa-miR-221*, hsa-miR-222, hsa-miR-222*, hsa-miR-223, hsa-miR-223*,hsa-miR-224, hsa-miR-23a, hsa-miR-23a*, hsa-miR-23b, hsa-miR-23b*,hsa-miR-24, hsa-miR-24-1*, hsa-miR-24-2*, hsa-miR-25, hsa-miR-25*,hsa-miR-26a, hsa-miR-26a-1*, hsa-miR-26a-2*, hsa-miR-26b, hsa-miR-26b*,hsa-miR-27a, hsa-miR-27a*, hsa-miR-27b, hsa-miR-27b*, hsa-miR-28-3p,hsa-miR-28-5p, hsa-miR-296-3p, hsa-miR-296-5p, hsa-miR-297, hsa-miR-298,hsa-miR-299-3p, hsa-miR-299-5p, hsa-miR-29a, hsa-miR-29a*, hsa-miR-29b,hsa-miR-29b-1*, hsa-miR-29b-2*, hsa-miR-29c, hsa-miR-29c*, hsa-miR-300,hsa-miR-301a, hsa-miR-301b, hsa-miR-302a, hsa-miR-302a*, hsa-miR-302b,hsa-miR-302b*, hsa-miR-302c, hsa-miR-302c*, hsa-miR-302d, hsa-miR-302d*,hsa-miR-302e, hsa-miR-302f, hsa-miR-30a*, hsa-miR-30a*, hsa-miR-30h,hsa-miR-30b*, hsa-miR-30c, hsa-miR-30c-1*, hsa-miR-30c-2*, hsa-miR-30d,hsa-miR-30d*, hsa-miR-30e, hsa-miR-30c*, hsa-miR-31, hsa-miR-31*,hsa-miR-32, hsa-miR-32*, hsa-miR-320a, hsa-miR-320b, hsa-miR-320c,hsa-miR-320d, hsa-miR-323-3p, hsa-miR-323-5p, hsa-miR-324-3p,hsa-miR-324-5p, hsa-miR-325, hsa-miR-326, hsa-miR-328, hsa-miR-329,hsa-miR-330-3p, hsa-miR-330-5p, hsa-miR-331-3p, hsa-miR-331-5p,hsa-miR-335, hsa-miR-335*, hsa-miR-337-3p, hsa-miR-337-5p,hsa-miR-338-3p, hsa-miR-338-5p, hsa-miR-339-3p, hsa-miR-339-5p,hsa-miR-33a, hsa-miR-33a*, hsa-miR-33b, hsa-miR-33b*, hsa-miR-340,hsa-miR-340*, hsa-miR-342-3p, hsa-miR-342-5p, hsa-miR-345, hsa-miR-346,hsa-miR-34a, hsa-miR-34a*, hsa-miR-34b, hsa-miR-34b*, hsa-miR-34c-3p,hsa-miR-34c-5p, hsa-miR-361-3p, hsa-miR-361-5p, hsa-miR-362-3p,hsa-miR-362-5p, hsa-miR-363, hsa-miR-363*, hsa-miR-365, hsa-miR-367,hsa-miR-367*, hsa-miR-369-3p, hsa-miR-369-5p, hsa-miR-370,hsa-miR-371-3p, hsa-miR-371-5p, hsa-miR-372, hsa-miR-373, hsa-miR-373*,hsa-miR-374a, hsa-miR-374a*, hsa-miR-374b, hsa-miR-374b*, hsa-miR-375,hsa-miR-376a, hsa-miR-376a*-hsa-miR-376b, hsa-miR-376c, hsa-miR-377,hsa-miR-377*, hsa-miR-378, hsa-miR-378*, hsa-miR-379, hsa-miR-379*,hsa-miR-380, hsa-miR-380*, hsa-miR-381, hsa-miR-382, hsa-miR-383,hsa-miR-384, hsa-miR-409-3p, hsa-miR-409-5p, hsa-miR-410, hsa-miR-411,hsa-miR-411*, hsa-miR-412, hsa-miR-421, hsa-miR-422a, hsa-miR-423-3p,hsa-miR-423-5p, hsa-miR-424, hsa-miR-424*, hsa-miR-425, hsa-miR-425*,hsa-miR-429, hsa-miR-431, hsa-miR-431*, hsa-miR-432, hsa-miR-432*,hsa-miR-433, hsa-miR-448, hsa-miR-449a, hsa-miR-449b, hsa-miR-450a,hsa-miR-450b-3p, hsa-miR-450h-5p, hsa-miR-451, hsa-miR-452,hsa-miR-452*, hsa-miR-453, hsa-miR-454, hsa-miR-454*, hsa-miR-455-3p,hsa-miR-455-5p, hsa-miR-483-3p, hsa-miR-483-5p, hsa-miR-484,hsa-miR-485-3p, hsa-miR-485-5p, hsa-miR-486-3p, hsa-miR-486-5p,h-a-miR-487u, hsa-miR-487b, ha-miR-488, hsa-miR-488*, hsa-miR-489,h-a-miR-490-3p, hsa-miR-490-5p, hsa-miR-491-3p, hsa-miR-491-5p,hsa-miR-492, hsa-miR-493, hsa-miR-493*, hsa-miR-494, hsa-miR-495,hsa-miR-496, hsa-miR-497, hsa-miR-497*, hsa-miR-498, hsa-miR-499-3p,hsa-miR-499-5p, hsa-miR-500, hsa-miR-500*, hsa-miR-501-3p,hsa-miR-501-5p, hsa-miR-502-3p, hsa-miR-502-5p, hsa-miR-503,hsa-miR-504, hsa-miR-505, hsa-miR-505*, hsa-miR-506, hsa-miR-507,hsa-miR-508-3p, hsa-miR-508-5p, hsa-miR-509-3-5p, hsa-miR-509-3p,hsa-miR-509-5p, hsa-miR-510, hsa-miR-511, hsa-miR-512-3p,hsa-miR-512-5p, hsa-miR-513a-3p, hsa-miR-513u-5p, hsa-miR-5I3b,hsa-miR-513c, hsa-miR-514, hsa-miR-515-3p, hsa-miR-515-5p,hsa-miR-516a-3p, hsa-miR-516a-5p, hsa-miR-516b, hsa-miR-517*,hsa-miR-517a, hsa-miR-517b, hsa-miR-517c, hsa-miR-518a-3p,hsa-miR-518a-5p, hsa-miR-518b, hsa-miR-518c, hsa-miR-518e*,hsa-miR-518d-3p, hsa-miR-518d-5p, hsa-miR-518e, hsa-miR-518c*,hsa-miR-518f, hsa-miR-518f*, hsa-miR-519a, hsa-miR-519b-3p,hsa-miR-519c-3p, hsa-miR-519d, hsa-miR-519e, hsa-miR-519e*,hsa-miR-520a-3p, hsa-miR-520a-5p, hsa-miR-520b, hsa-miR-520c-3p,hsa-miR-520d-3p, hsa-miR-520d-5p, hsa-miR-520e, hsa-miR-520f,hsa-miR-520g, hsa-miR-520h, hsa-miR-521, hsa-miR-522, hsa-miR-523,hsa-miR-524-3p, hsa-miR-524-5p, hsa-miR-525-3p, hsa-miR-525-5p,hsa-miR-526b, hsa-miR-526b*, hsa-miR-532-3p, hsa-miR-532-5p,hsa-miR-539, hsa-miR-541, hsa-miR-541*, hsa-miR-542-3p, hsa-miR-542-5p,hsa-miR-543, hsa-miR-544, hsa-miR-545, hsa-miR-545*, hsa-miR-548a-3p,hsa-miR-548a-5p, hsa-miR-548b-3p, hsa-miR-548b-5p, hsa-miR-548c-3p,hsa-miR-548c-5p, hsa-miR-548d-3p, hsa-miR-548d-5p, hsa-miR-548c,hsa-miR-548f, hsa-miR-548g, hsa-miR-548h, hsa-miR-548i, hsa-miR-548j,hsa-miR-548k, hsa-miR-548l, hsa-miR-548m, hsa-miR-548n, hsa-miR-548o,hsa-miR-548p, hsa-miR-549, hsa-miR-550, hsa-miR-550*, hsa-miR-551a,hsa-miR-551b, hsa-miR-551b*, hsa-miR-552, hsa-miR-553, hsa-miR-554,hsa-miR-555, hsa-miR-556-3p, hsa-miR-556-5p, hsa-miR-557, hsa-miR-558,hsa-miR-559, hsa-miR-561, hsa-miR-562, hsa-miR-563, hsa-miR-564,hsa-miR-566, hsa-miR-567, hsa-miR-568, hsa-miR-569, hsa-miR-570,hsa-miR-571, hsa-miR-572, hsa-miR-573, hsa-miR-574-3p, hsa-miR-574-5p,hsa-miR-575, hsa-miR-576-3p, hsa-miR-576-5p, hsa-miR-577, hsa-miR-578,hsa-miR-579, hsa-miR-580, hsa-miR-581, hsa-miR-582-3p, hsa-miR-582-5p,hsa-miR-583, hsa-miR-584, hsa-miR-585, hsa-miR-586, hsa-miR-587,hsa-miR-588, hsa-miR-589, hsa-miR-589*, hsa-miR-590-3p, hsa-miR-590-5p,hsa-miR-591, hsa-miR-592, hsa-miR-593, hsa-miR-593*, hsa-miR-595,hsa-miR-596, hsa-miR-597, hsa-miR-598, hsa-miR-599, hsa-miR-600,hsa-miR-601, hsa-miR-602, hsa-miR-603, hsa-miR-604, hsa-miR-605,hsa-miR-606, hsa-miR-607, hsa-miR-608, hsa-miR-609, hsa-miR-610,hsa-miR-611, hsa-miR-612, hsa-miR-613, hsa-miR-614, hsa-miR-615-3p,hsa-miR-615-5p, hsa-miR-616, hsa-miR-616*, hsa-miR-617, hsa-miR-618,hsa-miR-619, hsa-miR-620, hsa-miR-62I, hsa-miR-622, hsa-miR-623,hsa-miR-624, hsa-miR-624*, hsa-miR-625, hsa-miR-625*, hsa-miR-626,hsa-miR-627, hsa-miR-628-3p, hsa-miR-628-5p, hsa-miR-629, hsa-miR-629*,hsa-miR-630, hsa-miR-631, hsa-miR-632, hsa-miR-633, hsa-miR-634,hsa-miR-635, hsa-miR-636, hsa-miR-637, hsa-miR-638, hsa-miR-639,hsa-miR-640, hsa-miR-641, hsa-miR-642, hsa-miR-643, hsa-miR-644,hsa-miR-645, hsa-miR-646, hsa-miR-647, hsa-miR-648, hsa-miR-649,hsa-miR-650, h % a-miR-651, hsa-miR-652, hsa-miR-653, hsa-miR-654-3p,hsa-miR-654-5p, hsa-miR-655, hsa-miR-656, hsa-miR-657, hsa-miR-658,hsa-miR-659, hsa-miR-660, hsa-miR-661, hsa-miR-662, hsa-miR-663,hsa-miR-663b, hsa-miR-664, hsa-miR-664*, hsa-miR-665, hsa-miR-668,hsa-miR-671-3p, hsa-miR-671-5p, hsa-miR-675, hsa-miR-7, hsa-miR-708,hsa-miR-708*, hsa-miR-7-1*, hsa-miR-7-2′*, hsa-miR-720, hsa-miR-744,hsa-miR-744*, hsa-miR-758, hsa-miR-760, hsa-miR-765, hsa-miR-766,hsa-miR-767-3p, hsa-miR-767-5p, hsa-miR-7 68-3p, hsa-miR-768-5p,hsa-miR-769-3p, hsa-miR-769-5p, hsa-miR-770-5p, hsa-miR-802,hsa-miR-873, hsa-miR-874, hsa-miR-875-3p, hsa-miR-875-5p,hsa-miR-876-3p, hsa-miR-876-5p, hsa-miR-877, hsa-miR-877*,hsa-miR-885-3p, hsa-miR-885-5p, hsa-miR-886-3p, hsa-miR-886-5p,hsa-miR-887, hsa-miR-888, hsa-miR-888*, hsa-miR-889, hsa-miR-890,hsa-miR-891a, hsa-miR-891 b, hsa-miR-892a, hsa-miR-892b, hsa-miR-9,hsa-miR-9*, hsa-miR-920, hsa-miR-921, hsa-miR-922, hsa-miR-923,hsa-miR-924, hsa-miR-92a, hsa-miR-92a-1*, hsa-miR-92a-2*, hsa-miR-92b,hsa-miR-92b*, hsa-miR-93, hsa-miR-93*, hsa-miR-933, hsa-miR-934,hsa-miR-935, hsa-miR-936, hsa-miR-937, hsa-miR-938, hsa-miR-939,hsa-miR-940, hsa-miR-941, hsa-miR-942, hsa-miR-943, hsa-miR-944,hsa-miR-95, hsa-miR-96, hsa-miR-96*, hsa-miR-98, hsa-miR-99a,hsa-miR-99a*, hsa-miR-99b, and hsa-miR-99b*.

In some embodiments, one or more binding sites of a construct asdescribed by the disclosure (e.g., recombinant nucleic acid, AAV vector,rAAV, etc.) de-targets transgene expression from a cell of the immunesystem (e.g., an antigen presenting cell (APC)). In some embodiments, anmiRNA that de-targets transgene expression from an immune cell (e.g., anantigen presenting cell) is referred to as an immune-associated miRNA.In some embodiments, an immune-associated miRNA is an miRNA expressed inimmune cells that exhibits at least a 2-fold, 3-fold, 4-fold, 5-fold,6-fold, 7-fold, 8-fold, 9-fold, 10-fold higher level of expression in animmune cell compared with a non-immune cell (e.g., a control cell, suchas a HeLa cell, HEK293 cell, mesenchymal cell, etc.). In someembodiments, the cell of the immune system (immune cell) in which theimmune-associated miRNA is expressed is a B cell, T cell, Killer T cell,Helper T cell, γδ T cell, dendritic cell, macrophage, monocyte, vascularendothelial cell, or other immune cell. In some embodiments, the cell ofthe immune system is a B cell expressing one or more of the followingmarkers: B220, BLAST-2 (EBVCS), Bu-1, CD19, CD20 (26), CD22, CD24, CD27,CD57, CD72, CD79a, CD79b, CD86, chB6, D8/17, FMC7, L26, M17, MUM-1,Pax-5 (BSAP), and PC47H. In some embodiments, the cell of the immunesystem is a T cell expressing one or more of the following markers:ART2, CD1a, CD1d, CD11b (Mae-1), CD134 (OX40), CD150, CD2, CD25(interleukin 2 receptor alpha), CD3, CD38, CD4, CD45RO, CD5, CD7, CD72,CD8, CRTAM, FOXP3, FT2, GPCA, HLA-DR, HML-1, HT23A, Leu-22, Ly-2,Ly-m22, MICG, MRC OX 8, MRC OX-22, OX40, PD-1 (Programmed death-1), RT6,TCR (T cell receptor), Thy-1 (CD90), and TSA-2 (Thymic shared Ag-2). Insome embodiments, an immune-associated miRNA is selected from: miR-15a,miR-16-1, miR-17, miR-18a, miR-19a, miR-19b-1, miR-20a, miR-21,miR-29a/b/c, miR-30b, miR-31, miR-34a, miR-92a-1, miR-106a, miR-125a/b,miR-142-3p, miR-146a, miR-150, miR-155, miR-181a, miR-223 and miR-424,miR-221, miR-222, let-7i, miR-148, and miR-152.

Recombinant AAV Vectors (rAAV Vectors)

“Recombinant AAV (rAAV) vectors” of the disclosure are typicallycomposed of, at a minimum, a transgene and its regulatory sequences, and5′ and 3′ AAV inverted terminal repeats (ITRs). It is this recombinantAAV vector which is packaged into a capsid protein and delivered to aselected target cell. In some embodiments, the transgene is a nucleicacid sequence, heterologous to the vector sequences, which encodes apolypeptide, protein, functional RNA molecule (e.g., gRNA) or other geneproduct, of interest. The nucleic acid coding sequence is operativelylinked to regulatory components in a manner which permits transgenetranscription, translation, and/or expression in a cell of a targettissue.

In some embodiments, the instant disclosure relates to a recombinant AAV(rAAV) vector comprising a nucleic acid sequence including a promoteroperably linked to a transgene, wherein the transgene encodes a MeCP2protein (e.g., MeCP2 isoform e1). In some embodiments, a rAAV vectorfurther comprises nucleic acid sequences encoding one or more AAVinverted terminal repeat sequences (ITRs), for example AAV2 ITRs. Insome embodiments, a rAAV vector further comprises nucleic acid sequencesencoding one or more AAV ITRs selected from the group consisting ofAAV3, AAV4, AAV5, and AAV6. In some embodiments, ITRs are artificialsequences that replace ITR function, for example as disclosed inWO/2016/172008.

The AAV sequences of the vector typically comprise the cis-acting 5′ and3′ inverted terminal repeat sequences (See, e.g., B. J. Carter, in“Handbook of Parvoviruses”, ed., P. Tijsser. CRC Press, pp. 155 168(1990)). The ITR sequences are about 145 bp in length. Preferably,substantially the entire sequences encoding the ITRs are used in themolecule, although some degree of minor modification of these sequencesis permissible. The ability to modify these ITR sequences is within theskill of the art. (See, e.g., texts such as Sambrook et al. “MolecularCloning. A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory,New York (1989); and K. Fisher et al., J Virol., 70:520 532 (1996)). Anexample of such a molecule employed in the present disclosure is a“cis-acting” plasmid containing the transgene, in which the selectedtransgene sequence and associated regulatory elements are flanked by the5′ and 3′ AAV ITR sequences. The AAV ITR sequences may be obtained fromany known AAV, including presently identified mammalian AAV types (e.g.,AAV2. AAV3, AAV4. AAV5, or AAV6 ITR sequences).

In addition to the major elements identified above for the recombinantAAV vector, the vector also includes control elements necessary whichare operably linked to the transgene in a manner which permits itstranscription, translation and/or expression in a cell transfected withthe plasmid vector or infected with the virus produced by thedisclosure. As used herein, “operably linked” sequences include bothexpression control sequences that are contiguous with the gene ofinterest and expression control sequences that act in trans or at adistance to control the gene of interest.

Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation (polyA) signals;sequences that stabilize cytoplasmic mRNA: sequences that enhancetranslation efficiency (i.e., Kozak consensus sequence); sequences thatenhance protein stability; and when desired, sequences that enhancesecretion of the encoded product. A great number of expression controlsequences, including promoters which are native, constitutive, inducibleand/or tissue-specific, are known in the art and may be utilized.

As used herein, a nucleic acid sequence (e.g., coding sequence) andregulatory sequences are said to be “operably” linked when they arecovalently linked in such a way as to place the expression ortranscription of the nucleic acid sequence under the influence orcontrol of the regulatory sequences. If it is desired that the nucleicacid sequences be translated into a functional protein, two DNAsequences are said to be operably linked if induction of a promoter inthe 5′ regulatory sequences results in the transcription of the codingsequence and if the nature of the linkage between the two DNA sequencesdoes not (1) result in the introduction of a frame-shift mutation, (2)interfere with the ability of the promoter region to direct thetranscription of the coding sequences, or (3) interfere with the abilityof the corresponding RNA transcript to be translated into a protein.Thus, a promoter region would be operably linked to a nucleic acidsequence if the promoter region were capable of effecting transcriptionof that DNA sequence such that the resulting transcript might betranslated into the desired protein or polypeptide. Similarly two ormore coding regions are operably linked when they are linked in such away that their transcription from a common promoter results in theexpression of two or more proteins having been translated in frame. Insome embodiments, operably linked coding sequences yield a fusionprotein. In some embodiments, operably linked coding sequences yield afunctional RNA (e.g., gRNA, miRNA).

For nucleic acids encoding proteins, a polyadenylation sequencegenerally is inserted following the transgene sequences and before the3′ AAV ITR sequence. A rAAV construct useful in the present disclosuremay also contain an intron, desirably located between thepromoter/enhancer sequence and the transgene. One possible intronsequence is derived from SV40, and is referred to as the SV-40 T intronsequence. Another vector element that may be used is an internalribosome entry site (IRES). An IRES sequence is used to produce morethan one polypeptide from a single gene transcript. An IRES sequencewould be used to produce a protein that contain more than onepolypeptide chains. Selection of these and/or other vector elements maybe performed, as appropriate, and many such sequences are available[see, e.g., Sambrook et al. and references cited therein at, forexample, pages 3.18 3.26 and 16.17 16.27 and Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley & Sons, New York, 1989]. Insome embodiments, a Foot and Mouth Disease Virus 2A sequence is includedin polyprotein: this is a small peptide (approximately 18 amino acids inlength) that has been shown to mediate the cleavage of polyproteins(Ryan, M D et aL, EMBO, 1994; 4: 928-933; Mattion, N M et al., JVirology, November 1996; p. 8124-8127; Furler, S et al., Gene Therapy,2001: 8: 864-873: and Halpin. C et al., The Plant Journal, 1999; 4:453-459). The cleavage activity of the 2A sequence has previously beendemonstrated in artificial systems including plasmids and gene therapyvectors (AAV and retroviruses) (Ryan. M D et al., EMBO, 1994; 4:928-933; Mattion. N M et al., J Virology, November 1996; p. 8124-8127;Furler, S et al., Gene Therapy, 2001: 8: 864-873: and Halpin, C et al.,The Plant Journal, 1999; 4: 453-459; de Felipe, P et al., Gene Therapy,1999; 6: 198-208: de Felipe, P et al., Human Gene Therapy, 2000: 11:1921-1931.; and Klump, H et al., Gene Therapy, 2001: 8: 811-817).

The precise nature of the regulatory sequences needed for geneexpression in host cells may vary between species, tissues or celltypes, but shall in general include, as necessary, 5′ non-transcribedand 5′ non-translated sequences involved with the initiation oftranscription and translation respectively, such as a TATA box, cappingsequence, CAAT sequence, enhancer elements, and the like. Especially,such 5′ non-transcribed regulatory sequences will include a promoterregion that includes a promoter sequence for transcriptional control ofthe operably joined gene. Regulatory sequences may also include enhancersequences or upstream activator sequences as desired. The vectors of thedisclosure may optionally include 5′ leader or signal sequences. Thechoice and design of an appropriate vector is within the ability anddiscretion of one of ordinary skill in the art.

Examples of constitutive promoters include, without limitation, theretroviral Rous sarcoma virus (RSV) LTR promoter (optionally with theRSV enhancer), the cytomegalovirus (CMV) promoter (optionally with theCMV enhancer) [see. e.g., Boshart et al, Cell, 41:521-530 (1985)], theSV40 promoter, the dihydrofolate reductase promoter, the β-actinpromoter, the phosphoglycerol kinase (PGK) promoter, and the EF1αpromoter [Invitrogen]. In some embodiments, a promoter is an enhancedchicken β-actin promoter.

Inducible promoters allow regulation of gene expression and can beregulated by exogenously supplied compounds, environmental factors suchas temperature, or the presence of a specific physiological state, e.g.,acute phase, a particular differentiation state of the cell, or inreplicating cells only. Inducible promoters and inducible systems areavailable from a variety of commercial sources, including, withoutlimitation, Invitrogen, Clontech and Ariad. Many other systems have beendescribed and can be readily selected by one of skill in the art.Examples of inducible promoters regulated by exogenously suppliedpromoters include the zinc-inducible sheep metallothionine (MT)promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus(MMTV) promoter, the T7 polymerase promoter system (WO 98/10088); theecdysone insect promoter (No et al, Proc. Natl. Acad. Sci. USA,93:3346-3351 (1996)), the tetracycline-repressible system (Gossen et al,Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)), thetetracycline-inducible system (Gossen et al. Science, 268:1766-1769(1995), see also Harvey et al, Curr. Opin. Chem. Biol., 2:512-518(1998)), the RU486-inducible system (Wang et al, Nat. Biotech.,15:239-243 (1997) and Wang et al, Gene Ther., 4:432-441 (1997)) and therapamycin-inducible system (Magari et al, J. Clin. Invest.,100:2865-2872 (1997)). Still other types of inducible promoters whichmay be useful in this context are those which are regulated by aspecific physiological state, e.g., temperature, acute phase, aparticular differentiation state of the cell, or in replicating cellsonly.

In another embodiment, the native promoter for the transgene will beused. The native promoter may be preferred when it is desired thatexpression of the transgene should mimic the native expression. Thenative promoter may be used when expression of the transgene must beregulated temporally or developmentally, or in a tissue-specific manner,or in response to specific transcriptional stimuli. For example, in someembodiments, a native promoter is a MeCP2 promoter, such as a mouseMeCP2 promoter. In some embodiments, a mouse MeCP2 promoter isrepresented by a sequence set forth in SEQ ID NO: 3. In a furtherembodiment, other native expression control elements, such as enhancerelements, polyadenylation sites or Kozak consensus sequences may also beused to mimic the native expression.

In some embodiments, the regulatory sequences impart tissue-specificgene expression capabilities. In some cases, the tissue-specificregulatory sequences bind tissue-specific transcription factors, thatinduce transcription in a tissue specific manner. Such tissue-specificregulatory sequences (e.g., promoters, enhancers, etc.,) are well knownin the art. Exemplary tissue-specific regulatory sequences include, butare not limited to the following tissue specific promoters: aneye-specific retinoschisin promoter or K12 promoter, a liver-specificthyroxin binding globulin (TBG) promoter, an insulin promoter, aglucagon promoter, a somatostatin promoter, a pancreatic polypeptide(PPY) promoter, a synapsin-1 (Syn) promoter, a creatine kinase (MCK)promoter, a mammalian desmin (DES) promoter, a α-myosin heavy chain(a-MHC) promoter, or a cardiac Troponin T (cTnT) promoter. Otherexemplary promoters include Beta-actin promoter, hepatitis B virus corepromoter. Sandig et al., Gene Ther., 3:1002-9 (1996); alpha-fetoprotein(AFP) promoter, Arbuthnot et al., Hum. Gene Ther., 7:1503-14 (1996)),bone osteocalcin promoter (Stein et al., Mol. Biol. Rep., 24:185-96(1997)); bone sialoprotein promoter (Chen et al., J. Bone Miner. Res.,11:654-64 (1996)), CD2 promoter (Hansal et al., J. Immunol., 161:1063-8(1998); immunoglobulin heavy chain promoter: T cell receptor α-chainpromoter, neuronal such as neuron-specific enolase (NSE) promoter(Andersen et at, Cell. Mol. Neurobiol., 13:503-15 (1993)), neurofilamentlight-chain gene promoter (Piccioli et al., Proc. Natl. Acad. Sci. USA,88:5611-5 (1991)), and the neuron-specific vgf gene promoter (Piccioliet al., Neuron, 15:373-84 (1995)), among others which will be apparentto the skilled artisan.

In some embodiments, one or more bindings sites for one or more ofmiRNAs are incorporated in a transgene of a rAAV vector, to inhibit theexpression of the transgene in one or more tissues of an subjectharboring the transgene. The skilled artisan will appreciate thatbinding sites may be selected to control the expression of a transgenein a tissue specific manner. For example, binding sites for theliver-specific miR-122 may be incorporated into a transgene to inhibitexpression of that transgene in the liver. The target sites in the mRNAmay be in the 5′ UTR, the 3 UTR or in the coding region. Typically, thetarget site is in the 3′ UTR of the mRNA. Furthermore, the transgene maybe designed such that multiple miRNAs regulate the mRNA by recognizingthe same or multiple sites. The presence of multiple miRNA binding sitesmay result in the cooperative action of multiple RISCs and providehighly efficient inhibition of expression. The target site sequence maycomprise a total of 5-10), 10-60, or more nucleotides. The target sitesequence may comprise at least 5 nucleotides of the sequence of a targetgene binding site.

Recombinant Adeno-Associated Viruses (rAAVs)

In some aspects, the disclosure provides isolated AAVs. As used hereinwith respect to AAVs, the term “isolated” refers to an AAV that has beenartificially produced or obtained. Isolated AAVs may be produced usingrecombinant methods. Such AAVs are referred to herein as “recombinantAAVs”. Recombinant AAVs (rAAVs) preferably have tissue-specifictargeting capabilities, such that a nuclease and/or transgene of therAAV will be delivered specifically to one or more predeterminedtissue(s). The AAV capsid is an important element in determining thesetissue-specific targeting capabilities. Thus, an rAAV having a capsidappropriate for the tissue being targeted can be selected.

Methods for obtaining recombinant AAVs having a desired capsid proteinare well known in the art. (See, for example, US 2003/0138772), thecontents of which are incorporated herein by reference in theirentirety). Typically the methods involve culturing a host cell whichcontains a nucleic acid sequence encoding an AAV capsid protein; afunctional rep gene; a recombinant AAV vector composed of, AAV invertedterminal repeats (ITRs) and a transgene; and sufficient helper functionsto permit packaging of the recombinant AAV vector into the AAV capsidproteins. In some embodiments, capsid proteins are structural proteinsencoded by the cap gene of an AAV. AAVs comprise three capsid proteins,virion proteins 1 to 3 (named VP1, VP2 and VP3), all of which aretranscribed from a single cap gene via alternative splicing. In someembodiments, the molecular weights of VP1, VP2 and VP3 are respectivelyabout 87 kDa, about 72 kDa and about 62 kDa. In some embodiments, upontranslation, capsid proteins form a spherical 60-mer protein shellaround the viral genome. In some embodiments, the functions of thecapsid proteins are to protect the viral genome, deliver the genome andinteract with the host. In some aspects, capsid proteins deliver theviral genome to a host in a tissue specific manner.

In some embodiments, an rAAV described by the disclosure comprises oneor mom capsid proteins capable of crossing the blood-brain barrier. Insome embodiments, the at least one capsid protein has a serotypeselected from the group consisting of AAV1, AAV2, AAV2i8, AAV2.5, AAV6,AAV8, AAVrh8, AAV9, AAVrh10, AAV-B1, AAV9.45A-String (e.g., AAV9.45-AS),AAV9.45Angiopep, AAV9.47-Angiopep, and AAV9.47-AS. In some embodiments,the at least one capsid protein has a AAV-PHP.B serotype, for example asdescribed in U.S. Pat. No. 9,585,971. In some embodiments, a capsidprotein has a serotype as described in WO2015/127128, WO2016/054554,WO2016/054557, or WO2016/065001.

The components to be cultured in the host cell to package a rAAV vectorin an AAV capsid may be provided to the host cell in trans.Alternatively, any one or more of the required components (e.g.,recombinant AAV vector, rep sequences, cap sequences, and/or helperfunctions) may be provided by a stable host cell which has beenengineered to contain one or more of the required components usingmethods known to those of skill in the art. Most suitably, such a stablehost cell will contain the required component(s) under the control of aninducible promoter. However, the required component(s) may be under thecontrol of a constitutive promoter. Examples of suitable inducible andconstitutive promoters are provided herein, in the discussion ofregulatory elements suitable for use with the transgene. In stillanother alternative, a selected stable host cell may contain selectedcomponent(s) under the control of a constitutive promoter and otherselected component(s) under the control of one or more induciblepromoters. For example, a stable host cell may be generated which isderived from 293 cells (which contain E1 helper functions under thecontrol of a constitutive promoter), but which contain the rep and/orcap proteins under the control of inducible promoters. Still otherstable host cells may be generated by one of skill in the art.

In some embodiments, the instant disclosure relates to a host cellcontaining a nucleic acid that comprises a coding sequence encoding aprotein (e.g., MeCP2 protein, such as MeCP2 isoform e1). In someembodiments, the instant disclosure relates to a composition comprisingthe host cell described above. In some embodiments, the compositioncomprising the host cell above further comprises a cryopreservative.

The recombinant AAV vector, rep sequences, cap sequences, and helperfunctions required for producing the rAAV of the disclosure may bedelivered to the packaging host cell using any appropriate geneticelement (vector). The selected genetic element may be delivered by anysuitable method, including those described herein. The methods used toconstruct any embodiment of this disclosure are known to those withskill in nucleic acid manipulation and include genetic engineering,recombinant engineering, and synthetic techniques. See. e.g., Sambrooket al., Molecular Cloning: A Laboratory Manual, Cold Spring HarborPress, Cold Spring Harbor, N.Y. Similarly, methods of generating rAAVvirions are well known and the selection of a suitable method is not alimitation on the present disclosure. See. e.g., K. Fisher et al., J.Virol., 70:520-532 (1993) and U.S. Pat. No. 5,478,745.

In some embodiments, recombinant AAVs may be produced using the tripletransfection method (described in detail in U.S. Pat. No. 6,001,650).Typically, the recombinant AAVs are produced by transfecting a host cellwith an recombinant AAV vector (comprising a transgene) to be packagedinto AAV particles, an AAV helper function vector, and an accessoryfunction vector. An AAV helper function vector encodes the “AAV helperfunction” sequences (i.e., rep and cap), which function in trans forproductive AAV replication and encapsidation. Preferably, the AAV helperfunction vector supports efficient AAV vector production withoutgenerating any detectable wild-type AAV virions (i.e., AAV virionscontaining functional rep and cap genes). Non-limiting examples ofvectors suitable for use with the present disclosure include pHLP19,described in U.S. Pat. No. 6,001,650 and pRep6cap6 vector, described inU.S. Pat. No. 6,156,303, the entirety of both incorporated by referenceherein. The accessory function vector encodes nucleotide sequences fornon-AAV derived viral and/or cellular functions upon which AAV isdependent for replication (i.e., “accessory functions”). The accessoryfunctions include those functions required for AAV replication,including, without limitation, those moieties involved in activation ofAAV gene transcription, stage specific AAV mRNA splicing, AAV DNAreplication, synthesis of cap expression products, and AAV capsidassembly. Viral-based accessory functions can be derived from any of theknown helper viruses such as adenovirus, herpesvirus (other than herpessimplex virus type-1), and vaccinia virus.

In some aspects, the disclosure provides transfected host cells. Theterm “transfection” is used to refer to the uptake of foreign DNA by acell, and a cell has been “transfected” when exogenous DNA has beenintroduced inside the cell membrane. A number of transfection techniquesare generally known in the art. See, e.g., Giaham et al. (1973)Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratorymanual, Cold Spring Harbor Laboratories, New York. Davis et al. (1986)Basic Methods in Molecular Biology, Elsevier. and Chu et al. (1981) Gene13:197. Such techniques can be used to introduce one or more exogenousnucleic acids, such as a nucleotide integration vector and other nucleicacid molecules, into suitable host cells.

A “host cell” refers to any cell that harbors, or is capable ofharboring, a substance of interest. Often a host cell is a mammaliancell. A host cell may be used as a recipient of an AAV helper construct,an AAV minigene plasmid, an accessory function vector, or other transferDNA associated with the production of recombinant AAVs. The termincludes the progeny of the original cell which has been transfected.Thus, a “host cell” as used herein may refer to a cell which has beentransfected with an exogenous DNA sequence. It is understood that theprogeny of a single parental cell may not necessarily be completelyidentical in morphology or in genomic or total DNA complement as theoriginal parent, due to natural, accidental, or deliberate mutation.

As used herein, the term “cell line” refers to a population of cellscapable of continuous or prolonged growth and division in vitro. Often,cell lines are clonal populations derived from a single progenitor cell.It is further known in the art that spontaneous or induced changes canoccur in karyotype during storage or transfer of such clonalpopulations. Therefore, cells derived from the cell line referred to maynot be precisely identical to the ancestral cells or cultures, and thecell line referred to includes such variants.

As used herein, the terms “recombinant cell” refers to a cell into whichan exogenous DNA segment, such as DNA segment that leads to thetranscription of a biologically-active polypeptide or production of abiologically active nucleic acid such as an RNA, has been introduced.

As used herein, the term “vector” includes any genetic element, such asa plasmid, phage, transposon, cosmid, chromosome, artificial chromosome,virus, virion, etc., which is capable of replication when associatedwith the proper control elements and which can transfer gene sequencesbetween cells. Thus, the term includes cloning and expression vehicles,as well as viral vectors. In some embodiments, useful vectors arecontemplated to be those vectors in which the nucleic acid segment to betranscribed is positioned under the transcriptional control of apromoter. A “promoter” refers to a DNA sequence recognized by thesynthetic machinery of the cell, or introduced synthetic machinery,required to initiate the specific transcription of a gene. The phrases“operatively positioned,” “under control” or “under transcriptionalcontrol” means that the promoter is in the correct location andorientation in relation to the nucleic acid to control RNA polymeraseinitiation and expression of the gene. The term “expression vector orconstruct” means any type of genetic construct containing a nucleic acidin which part or all of the nucleic acid encoding sequence is capable ofbeing transcribed. In some embodiments, expression includestranscription of the nucleic acid, for example, to generate abiologically-active polypeptide product or functional RNA (e.g., guideRNA) from a transcribed gene. The foregoing methods for packagingrecombinant vectors in desired AAV capsids to produce the rAAVs of thedisclosure are not meant to be limiting and other suitable methods willbe apparent to the skilled artisan.

Administration Methods

Compositions described by the disclosure (e.g., recombinant nucleicacids, rAAVs, pharmaceutical compositions, etc.) may be delivered to asubject according to any appropriate methods known in the art.Compositions (e.g., recombinant nucleic acids, rAAVs, pharmaceuticalcompositions, etc.), preferably suspended in a physiologicallycompatible carrier (i.e., in a composition), may be administered to asubject, i.e. host animal, such as a human, mouse, rat, cat, dog, sheep,rabbit, horse, cow, goat, pig, guinea pig, hamster, chicken, turkey, ora non-human primate (e.g., Macaque). In some embodiments, a host animaldoes not include a human. In some embodiments, a subject is a human. Insome embodiments, a subject is less than a year old, for example 1, 2,3, 4, 5, 6, 7, 8, 9, 10, or 11 months old.

Delivery of the compositions to a mammalian subject may be by, forexample, systemic injection (e.g., intravenous injection) or intrathecalinjection. Additional methods of administering compositions to the CNSof a subject, for example intracranial injection, intrastriatalinjection, etc. may also be used. Combinations of administration methods(e.g., topical administration and intrastromal injection) can also beused.

In some embodiments, the compositions of the disclosure may comprise anrAAV alone, or in combination with one or more other viruses (e.g., asecond rAAV encoding having one or more different transgenes). In someembodiments, a composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore different rAAVs each having one or more different transgenes.

In some embodiments, a composition further comprises a pharmaceuticallyacceptable carrier. Suitable carriers may be readily selected by one ofskill in the art in view of the indication for which the composition isdirected. For example, one suitable carrier includes saline, which maybe formulated with a variety of buffering solutions (e.g., phosphatebuffered saline). Other exemplary carriers include sterile saline,lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin,peanut oil, sesame oil, and water. The selection of the carrier is not alimitation of the present disclosure.

Optionally, the compositions of the disclosure may contain, in additionto the recombinant nucleic acid or rAAV and carrier(s), otherpharmaceutical ingredients, such as preservatives, or chemicalstabilizers. Suitable exemplary preservatives include chlorobutanol,potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, theparabens, ethyl vanillin, glycerin, phenol, and parachlorophenol.Suitable chemical stabilizers include gelatin and albumin.

The compositions are administered in sufficient amounts to transfect thecells of a desired tissue (e.g., CNS tissue) and to provide sufficientlevels of gene transfer and expression without undue adverse effects.Examples of pharmaceutically acceptable mutes of administration include,but am not limited to, direct delivery to the selected organ (e.g.,intrastromal delivery to the eye), oral, inhalation (includingintranasal and intratracheal delivery), intraocular, intravenous,intramuscular, subcutaneous, intradermal, intratumoral, and otherparental routes of administration. Routes of administration may becombined, if desired.

The dose of rAAV virions required to achieve a particular “therapeuticeffect,” e.g., the units of dose in genome copies/per kilogram of bodyweight (GC/kg), will vary based on several factors including, but notlimited to: the route of rAAV virion administration, the level of geneor RNA expression required to achieve a therapeutic effect, the specificdisease or disorder being treated, and the stability of the gene or RNAproduct. One of skill in the art can readily determine a rAAV viriondose range to treat a patient having a particular disease or disorderbased on the aforementioned factors, as well as other factor.

An effective amount of composition (e.g., recombinant nucleic acid,rAAV, pharmaceutical composition, etc.) is an amount sufficient totarget infect an animal, target a desired tissue. In some embodiments,an effective amount of an rAAV is an amount sufficient to produce astable somatic transgenic animal model. The effective amount will dependprimarily on factors such as the species, age, weight, health of thesubject, and the tissue to be targeted, and may thus vary among animaland tissue. For example, an effective amount of the rAAV is generally inthe range of from about 1 ml to about 100 ml of solution containing fromabout 10⁹ to 10¹⁶ genome copies. In some cases, a dosage between about10¹¹ to 10¹³ rAAV genome copies is appropriate. In certain embodiments,10¹⁰ or 10¹¹ rAAV genome copies is effective to target CNS tissue (e.g.,corneal tissue). In some cases, stable transgenic animals are producedby multiple doses of an rAAV.

In some embodiments, a dose of the composition is administered to asubject no more than once per calendar day (e.g., a 24-hour period). Insome embodiments, a dose of the composition is administered to a subjectno more than once per 3, 4, 5, 6, or 7 calendar days. In someembodiments, a dose of the composition is administered to a subject nomore than once per calendar week (e.g., 7 calendar days). In someembodiments, a dose of the composition is administered to a subject nomore than bi-weekly (e.g., once in a two calendar week period). In someembodiments, a dose of the composition is administered to a subject nomore than once per calendar month (e.g., once in 30 calendar days). Insome embodiments, a dose of the composition is administered to a subjectno more than once per six calendar months. In some embodiments, a doseof the composition is administered to a subject no more than once percalendar year (e.g., 365 days or 366 days in a leap year).

In some embodiments, compositions are formulated to reduce aggregationof AAV particles in the composition, particularly where high rAAVconcentrations are present (e.g., ˜10¹³ GC/ml or more). Appropriatemethods for reducing aggregation of may be used, including, for example,addition of surfactants, pH adjustment, salt concentration adjustment,etc. (See, e.g., Wright F R, et al., Molecular Therapy (2005) 12,171-178, the contents of which are incorporated herein by reference.)

Formulation of pharmaceutically-acceptable excipients and carriersolutions is well-known to those of skill in the art, as is thedevelopment of suitable dosing and treatment regimens for using theparticular compositions described herein in a variety of treatmentregimens. Typically, these formulations may contain at least about 0.1%of the active compound or more, although the percentage of the activeingredient(s) may, of course, be varied and may conveniently be betweenabout 1 or 2% and about 70% or 80% or more of the weight or volume ofthe total formulation. Naturally, the amount of active compound in eachtherapeutically-useful composition may be prepared is such a way that asuitable dosage will be obtained in any given unit dose of the compound.Factors such as solubility, bioavailability, biological half-life, routeof administration, product shelf life, as well as other pharmacologicalconsiderations will be contemplated by one skilled in the art ofpreparing such pharmaceutical formulations, and as such, a variety ofdosages and treatment regimens may be desirable.

In some embodiments, compositions (e.g., recombinant nucleic acids,rAAVs, pharmaceutical compositions, etc.) in suitably formulatedpharmaceutical compositions disclosed herein are delivered directly totarget tissue, e.g., direct to CNS tissue (e.g., brain, spinal cord,etc.) However, in certain circumstances it may be desirable toseparately or in addition deliver the compositions via another route,e.g., subcutaneously, intraopancreatically, intranasally, parenterally,intravenously, intramuscularly, intrathecally, or orally,intraperitoneally, or by inhalation. In some embodiments, theadministration modalities as described in U.S. Pat. Nos. 5,543,158;5,641,515 and 5,399,363 (each specifically incorporated herein byreference in its entirety) may be used to deliver rAAVs. In someembodiments, a preferred mode of administration is by intravenousinjection.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. Dispersions may also be prepared in glycerol, liquidpolyethylene glycols, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations contain a preservativeto prevent the growth of microorganisms. In many cases the form issterile and fluid to the extent that easy syringability exists. It mustbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (e.g., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

For administration of an injectable aqueous solution, for example, thesolution may be suitably buffered, if necessary, and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, a suitable sterile aqueous medium may be employed. Forexample, one dosage may be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of the host.The person responsible for administration will, in any event, determinethe appropriate dose for the individual host.

Sterile injectable solutions am prepared by incorporating thecompositions (e.g., recombinant nucleic acids, rAAVs, pharmaceuticalcompositions, etc.) in the required amount in the appropriate solventwith various of the other ingredients enumerated herein, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the various sterilized active ingredients into asterile vehicle which contains the basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The compositions (e.g., recombinant nucleic acids, rAAVs, pharmaceuticalcompositions, etc.) disclosed herein may also be formulated in a neutralor salt form. Pharmaceutically-acceptable salts, include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike. Upon formulation, solutions will be administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective. The formulations are easily administered in avariety of dosage forms such as injectable solutions, drug-releasecapsules, and the like.

As used herein “carrier” includes any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Supplementary active ingredients can also be incorporated into thecompositions. The phrase “pharmaceutically-acceptable” refers tomolecular entities and compositions that do not produce an allergic orsimilar untoward reaction when administered to a host.

Delivery vehicles such as liposomes, nanocapsules, microparticles,microspheres, lipid particles, vesicles, and the like, may be used forthe introduction of the compositions of the present disclosure intosuitable host cells. In particular, the rAAV vector delivered transgenesmay be formulated for delivery either encapsulated in a lipid particle,a liposome, a vesicle, a nanosphere, or a nanoparticle or the like.

Such formulations may be preferred for the introduction ofpharmaceutically acceptable formulations of the nucleic acids or therAAV constructs disclosed herein. The formation and use of liposomes isgenerally known to those of skill in the art. Recently, liposomes weredeveloped with improved serum stability and circulation half-times (U.S.Pat. No. 5,741,516). Further, various methods of liposome and liposomelike preparations as potential drug carriers have been described (U.S.Pat. Nos. 5,567,434; 5,552,157; 5,565,213; 5,738,868 and 5,795,587).

Liposomes have been used successfully with a number of cell types thatare normally resistant to transfection by other procedures. In addition,liposomes are free of the DNA length constraints that are typical ofviral-based delivery systems. Liposomes have been used effectively tointroduce genes, drugs, radiotherapeutic agents, viruses, transcriptionfactors and allosteric effectors into a variety of cultured cell linesand animals. In addition, several successful clinical trials examiningthe effectiveness of liposome-mediated drug delivery have beencompleted.

Liposomes are formed from phospholipids that are dispersed in an aqueousmedium and spontaneously form multilamellar concentric bilayer vesicles(also termed multilarmellar vesicles (MLVs). MLVs generally havediameters of from 25 nm to 4 μm. Sonication of MLVs results in theformation of small unilamellar vesicles (SUVs) with diameters in therange of 200 to 500.ANG., containing an aqueous solution in the core.

Alternatively, nanocapsule formulations of the rAAV may be used.Nanocapsules can generally entrap substances in a stable andreproducible way. To avoid side effects due to intracellular polymericoverloading, such ultrafine particles (sized around 0.1 μm) should bedesigned using polymers able to be degraded in vivo. Biodegradablepolyalkyl-cyanoacrylate nanoparticles that meet these requirements arecontemplated for use.

Methods for Treating Rett Syndrome

In some aspects, the disclosure relates to compositions and methods fortreating Rett Syndrome. Rett syndrome is a genetic neurological disordercaused by one or more loss of function mutations in the MeCP2 gene, forexample as described in Suter et al. J Autism Dev Disord. 2014 March,44(3): 703-711. In some embodiments, a subject having Rett syndrome has1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more loss of function mutations inMeCP2 gene.

As used herein, the terms “treatment”, “treating”, and “therapy” referto therapeutic treatment and prophylactic or preventative manipulations.The terms further include ameliorating existing symptoms, preventingadditional symptoms, ameliorating or preventing the underlying causes ofsymptoms, preventing or reversing causes of symptoms, for example,symptoms associated with a disease caused by a loss of functionmutation, for example Rett syndrome. Thus, the terms denote that abeneficial result has been conferred on a subject with a disorder (e.g.,Rett syndrome), or with the potential to develop such a disorder.Furthermore, the term “treatment” is defined as the application oradministration of an agent (e.g., therapeutic agent or a therapeuticcomposition) to a subject, or an isolated tissue or cell line from asubject, who may have a disease, a symptom of disease or apredisposition toward a disease, with the purpose to cure, heal,alleviate, relieve, alter, remedy, ameliorate, improve or affect thedisease, the symptoms of disease or the predisposition toward disease.

Therapeutic agents or therapeutic compositions may include a compound ina pharmaceutically acceptable form that prevents and/or reduces thesymptoms of a particular disease (e.g., Rett syndrome). For example atherapeutic composition may be a pharmaceutical composition thatprevents and/or reduces the symptoms of Rett syndrome. It iscontemplated that the therapeutic composition of the present inventionwill be provided in any suitable form. The form of the therapeuticcomposition will depend on a number of factors, including the mode ofadministration as described herein. The therapeutic composition maycontain diluents, adjuvants and excipients, among other ingredients asdescribed herein.

EXAMPLE Example 1: Gene Expression Analysis of Human MeCP2 Isoform e1 InVitro

Approximately 80% of Rett cases are caused by mutations in the X-linkedgene encoding methyl CpG binding protein 2 (MeCP2), a widely expressedepigenetic regulator that is expressed at high levels in mature neurons.Most Rett patients carry a normal and mutant allele of MeCP2. Diseaseresults from random X-chromosome inactivation where ˜50% of neurons areMeCP2 deficient due to inactivation of the normal allele, whereas in theother ˜50% of neurons the mutant allele is silenced and normalexpression of wild type MeCP2is retained. The heterogeneity of MeCP2deficiency in the CNS has important implication, for development of genetherapy approaches for Rett syndrome. In Rett mouse models, thereversibility of neurological phenotypes has been observed afterrestoration of normal MeCP2 expression in adults. In these transgenicexperiments, restored MeCP2 expression was driven from its nativegenomic locus and activation was achieved in the majority of cells inthe brain. However, somatic gene transfer has yet to replicate any ofthese successes.

Generally, MeCP2 has a very narrow window of safe expression levels, aspatients with a duplication of the MeCP2 locus typically present delayedmotor and cognitive development as well as severe intellectualimpairment. Experiments in transgenic mouse models corroborate thisnotion, as ectopic expression of MeCP2 is toxic in wild-type animals,but safe and partially effective in ameliorating disease phenotypes ofMeCP2-deficient mice when transgene expression starts during embryonicdevelopment. Notably, the MeCP2 gene is alternatively spliced togenerate two proteins with different N termini, designated as MeCP2-e1and MeCP2-e2. Patients with MeCP2 locus duplication overexpress bothMECP2 isoforms. Therefore, the symptoms in patients with MeCP2 locusduplication and results in transgenic mice may be explained byoverexpression of the MeCP2-e2 isoform and timing of transgeneexpression during development.

Previous AAV9-MeCP2-e1 therapeutic experiments have been focused onneonatal intravascular (IV) or intracerebroventricular (ICV) deliveryand in some instances have encountered lethal liver toxicity and hindlimb clasping. Furthermore, the age of mice treated in such experimentsdoes not necessarily correspond to that likely to be implemented in mostRett patients, which presumably would be treated after symptom onset(6-18 months). In humans, the primary phase of synaptogenesis occurs inthe first 2 years and coincides with a rapid increase in non-CG DNAmethylation in neurons, as well as the onset of symptoms in Rettpatients. In mice, synaptogenesis occurs between 2 and 4 weeks of age.Therefore, it is critical to examine efficacy and potential toxicity ofAAV-MeCP2 gene delivery at relevant developmental stages beyondpost-natal day 0-1. Additionally, an important limitation toimplementing systemic AAV gene delivery to treat CNS disorders is thetransduction of organs other than the brain, such as liver, which is theorgan with the highest AAV tropism in the body.

A series of new AAV-MeCP2 vectors that eliminate gene expression inperipheral organs and also self-regulate expression of MeCP2 weredesigned. Generally, MeCP2 mRNA carries either a short (1.8kb) or long(˜10kb) 3′UTR, with the latter being the preferential isoform expressionin brain. The MeCP2 mRNA constructs described in this example comprisean MeCP2 isoform-e1 protein coding sequence and several miRNA regulatoryelements (MREs). It was observed that translation of MeCP2 in the CNS isregulated by miR-132 through a homeostatic mechanism involving changesin brain derived neurotrophic factor (BNDF) levels in response to MeCP2expression (FIG. 1A). Based on this mechanism a series of AAV-MeCP2vectors with increasing numbers of the miR-132 MREs (e.g., miR-132binding sites) coupled to a fixed number of MREs for miR-1 and miR-122(e.g., 3×-miR-1 and 3×-mir-122 binding sites) to de-target AAV geneexpression from skeletal muscle and liver (FIG. 1B).

A series of in vitro experiments were carried out. Briefly, HEK293Tcells were transfected with 30,000 gc/cell of AAV2-MeCP2 for four days.FIGS. 2A-2B show effective expression of AAV2-MeCP2 in HEK293T cells, asmeasured by Western blot (IG. 2A) and normalized protein expressionassay (FIG. 2B). FIG. 2C shows a toxicity profile of 293T cellstransduced with AAV2-MeCP2 for four days at a dose of 30,000 gc/cell.

A dose response study in mouse primary cortical neurons showedcomparable effects on cell survival for AAV-GFP and AAV-MeCP2 vectors(FIG. 3A), indicating that expression of myc-tagged human MeCP2 from ashort mouse MeCP2 promoter (−223 to +56) is non-toxic to primary neuronsin culture. In addition, it was observed that MeCP2-myc protein levelswere inversely proportional to the number of miR-132 MREs (e.g., miR-132binding sites) present in the MeCP2-myc transcript (FIG. 3B). FIG. 3Cshows miR-132 expression in response to AAV2-MeCP2 five days after AAVinfection.

Example 2: Gene Expression Analysis of Human MeCP2 Isoform e1 inWild-Type Mice Following Systemic Delivery of AAV-MeCP2

To extend the in vitro observations demonstrating the ability to titerMeCP2 levels by insertion of miR-132 target sequences described inExample 1, post-natal day 1 wild-type mice were injected via the facialvein (e.g., intracranial injection) with AAV encoding the e1 isoform ofhuman MeCP2 containing 0, 1×, 2×, or 3× miR-132 target sequences. Geneexpression analysis of brain tissue indicated that MeCP2 levels areinversely proportional to the number of miR132 target sequences (FIGS.4A-4C).

In some embodiments, systemic administration of some AAV serotypes cantransduce tissues outside of the central nervous system, and elevatedexpression of MeCP2 in liver, cardiac and skeletal tissues has beenobserved to be associated with detrimental physiological consequences.To minimize heart and liver transduction of MeCP2, AAV-MeCP2 vectorsdescribed by the disclosure contain at least one miR-1 (e.g., 3×-miR-1)and at least one miR-122 (e.g., 3×-miR-122) target sequence (e.g.,binding sites) to de-target MeCP2 expression from the heart and liver,respectively, qRT-PCR analysis using primers against e2 human MeCP2(which was undetectable), and e1 and e2 mouse MeCP2 (which did notchange) were performed. Gene expression analysis of heart and livertissue from wild-type animals indicated MeCP2 is effectively de-targetedfrom the heart and liver, as evidenced by substantially reducedexpression compared to the brain (FIG. 4A and FIG. 5 ).

Example 3: Therapeutic Efficacy and Safety of Self-Regulating AAV-MeCP2Vectors

Therapeutic efficacy and safety of AAV-MeCP2-e| vectors is examined inmice. In some embodiments, AAV-PHP.B capsid protein is used, as thiscapsid has improved neuronal transduction efficiency. Mecp2-null mice(Mecp2^(tm1.1Bind)/J; Male^(−/y) and female^(+/−)) at 4 weeks of age aretreated by systemic administration of AAV-PHP.B-MeCP2-e1 vectorscarrying different MRE cassettes (e.g., at vector doses of 1E11, 3E11,1E12 vg/mouse) and body weight and phenotypic scores are monitored everytwo weeks. As controls. MeCP2/Mecp2^(tm1.1Bind) mice injected withvehicle and wild type mice are used. A subset of mice in each cohort(n=8; 4 males and 4 females) are sacrificed at 8 weeks post-injectionand MeCP2 expression quantified by western blot and compared acrossgroups. Transduction efficiency in the brain is assessed by doubleimmunofluorescence staining for MeCP2 and neurons (using the neuronalmarker NeuN) and quantification of transduced neurons (MeCP2+, NeuN+) incortex, striatum, thalamus, hippocampus and cerebellum is performed. Thelevels of PSD-95 are assessed by western blot and immunostaining ofbrain sections: PSD-95 is a key scaffold protein in synaptic maturationwhose levels are decreased in brains from MeCP2-null mice.

To perform vector biodistribution analysis, genomic DNA is isolated fromdifferent regions of the CNS and peripheral organs and analyzed bydigital PCR. Another subset of animals in each cohort (n=16; 8 males and8 females) is used for survival and longitudinal analysis of behavioral(e.g., open field; social interaction) and motor performance (e.g.,rotarod, grid walk) as well as whole body plethismography to assessbreathing patterns and apnea characteristic of MeCP2-null mice. Endpointstudies are the same as at 8 weeks after treatment. Safety of thevectors is also assessed in wild type mice in a dose escalation studyusing doses identical to those indicated above with endpoints at 7, 30,90 and 180 days to assess the CNS and peripheral tissues for evidence oftoxicity. AAV vector biodistribution and MeCP2 expression are assessedas well.

Example 4: Characterization of Changes in the Genome/Transcriptome ofTransduced Neurons after AAV-MeCP2 Gene Transfer at Different Stages ofNervous System Development

A key aspect in the development of a safe and effective gene therapyapproach for Rett is to characterize in detail the impact of de novoexpression of MeCP2 on the epigenetic landscape and transcriptomicprofile of transduced neurons. For this purpose. AAV-PHP.B-MeCP2-e1vectors carrying an IRES-GFP cassette are produced, and allow isolationof transduced GFP+ cells from brain, cerebellum and spinal cord byeither FACS or laser capture microdissection followed by whole genomebisulfite sequencing (MethylC-Seq), small RNA-seq (microRNAs), andRNA-Seq (mRNAs and non-coding RNAs). MeCP2^(−/y) males. MeCP2^(−/+)females and wild-type controls (males and females) receive a systemicinjection of AAV-PHP.B-MeCP2-e1-IRES-GFP, control vector (without MeCP2cDNA) and vehicle at day 1, 7, 14, and 28, as well as at 12 weeks of ageat an optimal dose. Mice (n=8; 4 males and 4 females) are euthanized at1 or 3 months after injection to assess the parameters indicated above.Information on microRNAs that are overexpressed in response to MeCP2expression is used to established additional layers of gene expressionregulation in addition to that based on miR-132.

Example 5: Contribution of MeCP2 Isoforms to Therapeutic Success orOnset of Neurological Symptoms as a Function of Intervention atDifferent Stages of Development

MeCP2 expression of 1.6- to 6-fold above physiologically normal levelshas been observed to cause neurological symptoms both in patients withMeCP2 locus duplication (˜2-fold above normal) and transgenic mousemodels. The other commonality between patients and transgenic mousemodels is that both overexpress the MeCP2-e2 isoform, which unlike thee1 isoform appears to be toxic to primary neurons in culture. In someembodiments, this toxic effect is eliminated by co-expression of FoxG1,which is another gene where mutations are associated with Rett syndrome.In some embodiments, co-expression of FoxG1 with MeCP2 is an additionalmechanism to control the side effects associated with MeCP2overexpression. Therapeutic, safety and epigenomic/transcriptomicexperiments with AAV-PHP.B vectors encoding MeCP-e1, MeCP2-e2, MeCP2-e2and FoxG1, or FoxG1 alone in are conducted MeCP2^(−/y) males,MeCP2^(+/−) females and wild-type age matched controls.

SEQUENCES >SEQ ID NO: 1; human MeCP2 isoform el amino acid sequence (NM_001110792)MAAAAAAAPSGGGGGGBEERLEEKSEDQDLQGLKDKPLKFKKVKKDKKEEKEGKHEPVQPSAHHSAEPAEAGKAETSEGSGSAPAVPEASASPKQRRSIIRDRGPMYDDPTLPEGWTRKIKQRKSGRSAGKYDVYLINPQGKAFRSKVELIAYFEKVGDTSLDPNDFDFTVTGRGSPSRREQKPPKKFKSPKAPGTGRGRGRPKGSGTTRPKAATSEGVQVKRVLEKSPGKLLVKMPFQTSPGGKAEGGGATTSTQVMVIKRPGRKRKAEADPQAIPKKRGRKPGSVVAAAAAEAKKKAVKESSIRSVQETVLPIKKRKTRETVSIEVKEVVKPLLVSTLGEKSGKGLKTCKSPGRKSKESSPKGRSSSASSPPKKEHHHHHHHSESPKAPVPLLPPLPPPPPEPESSEDPTSPPEPQDLSSSVCKEEKMPRGGSLESDGCPKEPAKTQPAVATAATAAEKYKHRGEGERKDIVSSSMPRPNREEPVDSRTPVTERVS >SEQ ID NO: 2; human MeCP2 isoform e2 amino acid sequence (NM_004992)MVAGMLGLREEKSEDQDLQGLKDKPLKFKKVKKDKKEEKEGKHEPVQPSAHHSAEPAEAGKAETSEGSGSAPAVPEASASPKQRRSIIRDRGPMYDDPTLPEGWTRKLKQRKSGRSAGKYDVYLINPQGKAFRSKVELIAYFEKVGDTSLDPNDFDFTVTGRGSPSRREQKPPKKPKSPKAPGTGRGRGRPKGSGTTRPKAATSEGVAVKRVLEKSPGKLLVKMPFQTSPGGKAEGGGATTSTQVMVIKRPGRKRKAEADPQAIPKKRGRKPGSVVAAAAAEAKKKAVKESSIRSVQETVLPIKKRKTRETVSIEVKEVVKPLLVSTLGEKSGKGLKTCKSPGRKSKESSPKGRSSSASSPPKKEHHHHHHHSESPKAPVPLLPPLPPPPPEPESSEDPTSPPEPQDLSSSVCKEEKMPRGGSLESDGCPKEPAKTQPAVATAATAAEKYKHRGEGERKDIVSSSMPRPNREEPVDSRIPVTERVS >SEQ ID NO: 3; mouse MeCP2 promoter DNA sequenceAATTGAGGGCGTCACCGCTAAGGCTCCGCCCCAGCCTGGGCTCCACAACCAATGAAGGGTAATCTCGACAAAGAGCAAGGGGTGGGGCGCGGGCGCGCAGGTGCAGCAGCACACAGGCTGGTCGGGAGGGGGGGGCGCGACGTCTGCCGTGCGGGGTCCCGGCATCGGTT >SEQ ID NO: 4; miR-122 binding site DNA sequenceACAAACACCATTGTCACACTCCA >SEQ ID NO: 5; miR-1 binding site DNA sequenceATACATACTTCTTTACATTCCA >SEQ ID NO: 6; miR-132 binding site DNA sequenceCGACCATGGCTGTAGACTGTTA >SEQ ID NO: 7; MeCP2 in vitro construct nucleic acid sequence (scAAV-Mec229-hMeCP2-miR132(1x) miR122-1(3x) plasmid)CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGTAGCCATGCTCTAGGAAGATCAATTCGGTACAATTCACGCGTCGACAATTGAGGGCGTCACCGCTAAGGCTCCGCCCCAGCCTGGGCTCCACAACCAATGAAGGGTAATCTCGACAAAGAGCAAGGGGTGGGGCGCGGGCGCGCAGGTGCAGCAGCACACAGGCTGGTCGGGAGGGCGGGGCGCGACGTCTGCCGTGCGGGGTCCCGGCATCGGTTGCGCGCGCGCTCCCTCCTCTCGGAGAGAGGGCTGTGGTAAAACCCGTCCGGAAAATGGCTGCAGCCGCTGCCGCAGCGCCGAGCGGCGGAGGTGGCGGTGGCGAGGAGGAGAGACTGGAAGAAAAGTCAGAAGACCAGGACCTCCAGGGCCTCAAGGACAAACCCCTCAAGTTTAAAAAGGTGAAGAAAGATAAGAAAGAAGAGAAAGAGGGCAAGCATGAGCCCGTGCAGCCATCAGCCCACCACTCTGCTGAGCCCCCAGAGGCAGGCAAAGCAGAGACATCAGAAGGGTCAGGCTCCGCCCCGGCTGTGCCGGAAGCTTCTGCCTCCCCCAAACAGCGGCGCTCCATCATCCGTGACCGGGGACCCATGTATGATGACCCCACCCTGCCTGAAGGCTGGACACGGAAGCTTAAGCAAAGGAAATCTGGACGCTCTGCTGGGAAGTATGATGTGTATTTGATCAATCCCCAGGGAAAAGCCTTTCGCTCTAAAGTGGAGTTGATTGCGTACTTCGAAAAGGTAGGCGACACATCCCTGGACCCTAATGATTTTGACTTCACGGTAACTGGGAGAGGGAGCCCCTCCCGGCGAGAGCAGAAACCACCTAAGAAGCCCAAATCTCCCAAAGCTCCAGGAACTGGCAGAGGTCGGGGACGCCCCAAAGGGAGCGGCACCACGAGACCCAAGGCAGCTACGTCAGAGGGTGTGCAGGTGAAAAGGGTCCTGGAGAAAAGTCCTGGGAAGCTCCTTGTCAAGATGCCTTTTCAAACTTCGCCAGGGGGCAAGGCTGAGGGGGGTGGGGCCACCACATCCACCCAGGTCATGGTGATCAAACGCCCCGGCAGGAAGCGAAAAGCTGAGGCAGACCCTCAGGCCATTCCCAAGAAACGGGGTCGAAAGCCGGGGAGTGTGGTGGCAGCCGCTGCCGCCGAGGCCAAAAAGAAAGCCGTGAAGGAGTCTTCTATCCGATCTGTGCAGGAGACCGTACTCCCCATCAAGAAGCGCAAGACCCGGGAGACGGTCAGCATCGAGGTCAAGGAAGTGGTGAAGCCCCTGCTGGTGTCCACCCTCGGTGAGAAGAGCGGGAAAGGACTGAAGACCTGTAAGAGCCCTGGGCGGAAAAGCAAGGAGAGCAGCCCCAAGGGGCGCAGCAGCAGCGCCTCCTCACCCCCCAAGAAGGAGCACCACCACCATCACCACCACTCAGAGTCCCCAAAGGCCCCCGTGCCACTGCTCCCACCCCTGCCCCCACCTCCACCTGAGCCCGAGAGCTCCGAGGACCCCACCAGCCCCCCTGAGCCCCAGGACTTGAGCAGCAGCGTCTGCAAAGAGGAGAAGATGCCCAGAGGAGGCTCACTGGAGAGCGACGGCTGCCCCAAGGAGCCAGCTAAGACTCAGCCCGCGGTTGCCACCGCCGCCACGGCCGCAGAAAAGTACAAACACCGAGGGGAGGGAGAGCGCAAAGACATTGTTTCATCCTCCATGCCAAGGCCAAACAGAGAGGAGCCTGTGGACAGCCGGACGCCCGTGACCGAGAGAGTTAGCGAGCAGAAGCTGATCTCAGAGGAGGACCTGTGACGACCATGGCTGTAGACTGTTACTCGAGATACATACTTCTTTACATTCCAATACATACTTCTTTACATTCCAATACATACTTCTTTACATTCCACCATGGACTAGTACAAACACCATTGTCACACTCCAACAAACACCATTGTCACACTCCAACAAACACCATTGTCACACTCCAGCGGCCGCTTCGATCCGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGCCTAGGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACAAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACCCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCCTTAATTAACCTAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCCCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATGGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATGTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAGATTTAATTAAGGCCTTAATTAGG >SEQ ID NO: 8; MeCP2 in vitro construct nucleic acid sequence (scAAV-Mex229-hMeCP2-miR132(2x) miR122-1(3x) plasmid)CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGTAGCCATGCTCTAGGAAGATCAATTCGGTACAATTCACGCGTCGACAATTGAGGGCGTCACCGCTAAGGCTCCGCCCCAGCCTGGGCTCCACAACCAATGAAGGGTAATCTCGACAAAGAGCAAGGGGTGGGGCGCGGGCGCGCAGGTGCAGCAGCACACAGGCTGGTCGGGAGGGCGGGGCGCGACGTCTGCCGTGCGGGGTCCCGGCATCGGTTGCGCGCGCGCTCCCTCCTCTCGGAGAGAGGGCTGTGGTAAAACCCGTCCGGAAAATGGCTGCAGCCGCTGCCGCAGCGCCGAGCGGCGGAGGTGGCGGTGGCGAGGAGGAGAGACTGGAAGAAAAGTCAGAAGACCAGGACCTCCAGGGCCTCAAGGACAAACCCCTCAAGTTTAAAAAGGTGAAGAAAGATAAGAAAGAAGAGAAAGAGGGCAAGCATGAGCCCGTGCAGCCATCAGCCCACCACTCTGCTGAGCCCCCAGAGGCAGGCAAAGCAGAGACATCAGAAGGGTCAGGCTCCGCCCCGGCTGTGCCGGAAGCTTCTGCCTCCCCCAAACAGCGGCGCTCCATCATCCGTGACCGGGGACCCATGTATGATGACCCCACCCTGCCTGAAGGCTGGACACGGAAGCTTAAGCAAAGGAAATCTGGACGCTCTGCTGGGAAGTATGATGTGTATTTGATCAATCCCCAGGGAAAAGCCTTTCGCTCTAAAGTGGAGTTGATTGCGTACTTCGAAAAGGTAGGCGACACATCCCTGGACCCTAATGATTTTGACTTCACGGTAACTGGGAGAGGGAGCCCCTCCCGGCGAGAGCAGAAACCACCTAAGAAGCCCAAATCTCCCAAAGCTCCAGGAACTGGCAGAGGTCGGGGACGCCCCAAAGGGAGCGGCACCACGAGACCCAAGGCAGCTACGTCAGAGGGTGTGCAGGTGAAAAGGGTCCTGGAGAAAAGTCCTGGGAAGCTCCTTGTCAAGATGCCTTTTCAAACTTCGCCAGGGGGCAAGGCTGAGGGGGGTGGGGCCACCACATCCACCCAGGTCATGGTGATCAAACGCCCCGGCAGGAAGCGAAAAGCTGAGGCAGACCCTCAGGCCATTCCCAAGAAACGGGGTCGAAAGCCGGGGAGTGTGGTGGCAGCCGCTGCCGCCGAGGCCAAAAAGAAAGCCGTGAAGGAGTCTTCTATCCGATCTGTGCAGGAGACCGTACTCCCCATCAAGAAGCGCAAGACCCGGGAGACGGTCAGCATCGAGGTCAAGGAAGTGGTGAAGCCCCTGCTGGTGTCCACCCTCGGTGAGAAGAGCGGGAAAGGACTGAAGACCTGTAAGAGCCCTGGGCGGAAAAGCAAGGAGAGCAGCCCCAAGGGGCGCAGCAGCAGCGCCTCCTCACCCCCCAAGAAGGAGCACCACCACCATCACCACCACTCAGAGTCCCCAAAGGCCCCCGTGCCACTGCTCCCACCCCTGCCCCCACCTCCACCTGAGCCCGAGAGCTCCGAGGACCCCACCAGCCCCCCTGAGCCCCAGGACTTGAGCAGCAGCGTCTGCAAAGAGGAGAAGATGCCCAGAGGAGGCTCACTGGAGAGCGACGGCTGCCCCAAGGAGCCAGCTAAGACTCAGCCCGCGGTTGCCACCGCCGCCACGGCCGCAGAAAAGTACAAACACCGAGGGGAGGGAGAGCGCAAAGACATTGTTTCATCCTCCATGCCAAGGCCAAACAGAGAGGAGCCTGTGGACAGCCGGACGCCCGTGACCGAGAGAGTTAGCGAGCAGAAGCTGATCTCAGAGGAGGACCTGTGACGACCATGGCTGTAGACTGTTACGACCATGGCTGTAGACTGTTACTCGAGATACATACTTCTTTACATTCCAATACATACTTGTTTACATTCCAATACATACTTCTTTACATTCCACCATGGACTAGTACAAACACCATTGTCACACTCCAACAAACACCATTGTCACACTCCAACAAACACCATTGTCACACTCCAGCGGCCGCTTCGATCCGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGCCTAGGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACAAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACCCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCCTTAATTAACCTAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCACCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCGTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATGTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACACCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGGTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGGGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAGATTTAATTAAGGCCTTAATTAGG >SEQ ID NO: 9; MeCP2 in vitro construct nucleic acid sequence (scAAV-Mec229-hMeCP2-miR132(3x) miR122-1(3x) plasmid)CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGTAGCCATGGTCTAGGAAGATCAATTCGGTACAATTCACGCGTCGACAATTGAGGGCGTCACCGCTAAGGCTCCGCCCCAGCCTGGGCTCCACAACCAATGAAGGGTAATCTCGACAAAGAGCAAGGGGTGGGGCGCGGGCGCGCAGGTGCAGCAGCACACAGGCTGGTCGGGAGGGCGGGGCGCGACGTCTGCCGTGCGGGGTCCCGGCATCGGTTGCGCGCGCGCTCCCTCCTCTCGGAGAGAGGGCTGTGGTAAAACCCGTCCGGAAAATGGCTGCAGCCGCTGCCGCAGCGCCGAGCGGCGGAGGTGGCGGTGGCGAGGAGGAGAGACTGGAAGAAAAGTCAGAAGACCAGGACCTCCAGGGCCTCAAGGACAAACCCCTCAAGTTTAAAAAGGTGAAGAAAGATAAGAAAGAAGAGAAAGAGGGCAAGCATGAGCCCGTGCAGCCATCAGCCCACCACTCTGCTGAGCCCGCAGAGGCAGGCAAAGCAGAGACATCAGAAGGGTCAGGCTCCGCCCCGGCTGTGCCGGAAGCTTCTGCCTCCCCCAAACAGCGGCGCTCCATCATCCGTGACCGGGGACCCATGTATGATGACCCCACCCTGCCTGAAGGCTGGACACGGAAGCTTAAGCAAAGGAAATCTGGACGCTCTGCTGGGAAGTATGATGTGTATTTGATCAATCCCCAGGGAAAAGCCTTTCGCTCTAAAGTGGAGTTGATTGCGTACTTCGAAAAGGTAGGCGACACATCCCTGGACCCTAATGATTTTGACTTCACGGTAACTGGGAGAGGGAGCCCCTCCCGGCGAGAGCAGAAACCACCTAAGAAGCCCAAATCTCCCAAAGCTCCAGGAACTGGCAGAGGTCGGGGACGCCCCAAAGGGAGCGGCACCACGAGACCCAAGGCAGCTACGTCAGAGGGTGTGCAGGTGAAAAGGGTCCTGGAGAAAAGTCCTGGGAAGCTCCTTGTCAAGATGCCTTTTCAAACTTCGCCAGGGGGCAAGGCTGAGGGGGGTGGGGCCACCACATCCACCCAGGTCATGGTGATCAAACGCCCCGGCAGGAAGCGAAAAGCTGAGGCAGACCCTCAGGCCATTCCCAAGAAACGGGGTCGAAAGCCGGGGAGTGTGGTGGCAGCCGCTGCCGCCGAGGCCAAAAAGAAAGCCGTGAAGGAGTCTTCTATCCGATCTGTGCAGGAGACCGTACTCCCCATCAAGAAGCGCAAGACCCGGGAGACGGTCAGCATCGAGGTCAAGGAAGTGGTGAAGCCCCTGCTGGTGTCCACCCTCGGTGAGAAGAGCGGGAAAGGACTGAAGACCTGTAAGAGCCCTGGGCGGAAAAGCAAGGAGAGCAGCCCCAAGGGGCGCAGCAGCAGCGCCTCCTCACCCCCCAAGAAGGAGCACCACCACCATCACCACCACTCAGAGTCCCCAAAGGCCCCCGTGCCACTGCTCCCACCCCTGCCCCCACCTCCACCTGAGCCCGAGAGCTCCGAGGACCCCACCAGCCCCCCTGAGCCCCAGGACTTGAGCAGCAGCGTCTGCAAAGAGGAGAAGATGCCCAGAGGAGGCTCACTGGAGAGCGACGGCTGCCCCAAGGAGCCAGCTAAGACTCAGCCCGCGGTTGCCACCGCCGCCACGGCCGCAGAAAAGTACAAACACCGAGGGGAGGGAGAGCGCAAAGACATTGTTTCATCCTCCATGCCAAGGCCAAACAGAGAGGAGCCTGTGGACAGCCGGACGCCCGTGACCGAGAGAGTTAGCGAGCAGAAGCTGATCTCAGAGGAGGACCTGTGACGACCATGGCTGTAGACTGTTACGACCATGGCTGTAGACTGTTACCACCATGGCTGTAGACTGTTACTCGAGATACATACTTCTTTACATTCCAATACATACTTCTTTACATTCCAATACATACTTCTTTACATTCCACCATGGACTAGTACAAACACCATTGTCACACTCCAACAAACACCATTGTCACACTCCAACAAACACCATTGTCACACTCCAGCGGCCGCTTCGATCCGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGCCTAGGTAGATAAGTAGCATGGCGCGTTAATCATTAACTACAAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCCTTAATTAACCTAATTCACTGGCCGTCGTTTTACAACCTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGGGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATGTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAGATTTAATTAAGGCCTTAATTAGG >SEQ ID NO: 10; MeCP2 in vivo construct nucleic acid sequence (scAAV-Mec229-bMeCP2-miR132(1x) miR 122-1(3x) vector genome)CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGTAGCCATGCTCTAGGAAGATCAATTCGGTACAATTCACGCGTCGACAATTGAGGGCGTCACCGCTAAGGCTCCGCCCCAGCCTGGGCTCCACAACCAATGAAGGGTAATCTCGACAAAGAGCAAGGGGTGGGGCGCGGGCGCGCAGGTGCAGCAGCACACAGGCTGGTCGGGAGGGCGGGGCGCGACGTCTGCCGTGCGGGGTCCCGGCATCGGTTGCGCGCGCGCTCCCTCCTCTCGGAGAGAGGGCTGTGGTAAAACCCGTCCGGAAAATGGCTGCAGCCGCTGCCCCAGCGCCGAGCGGCGGAGGTGGCGGTGGCGAGGAGGAGAGACTGGAAGAAAAGTCAGAAGACCAGGACCTCCAGGGCCTCAAGGACAAACCCCTCAAGTTTAAAAAGGTGAAGAAAGATAAGAAAGAAGAGAAAGAGGGCAAGCATGAGCCCGTGCAGCCATCACCCCACCACTCTGCTGAGCCCGCAGAGGCAGGCAAAGCAGAGACATCAGAAGGGTCAGGCTCCGCCCCGGCTGTGCCGGAAGCTTCTGCCTCCCCCAAACAGCGGCGCTCCATCATCCGTGACCGGGGACCCATGTATGATGACCCCACCCTGCCTGAAGGCTGGACACGGAAGGTTAAGCAAAGGAAATCTGGACGCTCTGCTGGGAAGTATGATGTGTATTTGATCAATCCCCAGGGAAAAGCCTTTCGCTCTAAAGTGGAGTTGATTGCGTACTTCGAAAAGGTAGGCGACACATCCCTGGACCCTAATGATTTTGACTTCACGGTAACTGGGAGAGGGAGCCCCTCCCGGCGAGAGCAGAAACCACCTAAGAAGCCCAAATCTCCCAAAGCTCCAGGAACTGGCAGAGGTCGGGGACGCCCCAAAGGGAGCGGCACCACGAGACCCAAGGCAGCTACGTCAGAGGGTGTGCAGGTGAAAAGGGTCCTGGAGAAAAGTCCTGGGAAGCTCCTTGTCAAGATGCCTTTTCAAACTTCGCCAGGGGGCAAGGCTGAGGGGGGTGGGGCCACCACATCCACCCAGGTCATGGTGATCAAACGCCCCGGCAGGAAGCGAAAAGCTGAGGCAGACCCTCAGGCCATTCCCAAGAAACGGGGTCGAAAGCCGGGGAGTGTGGTGGCAGCCGCTGCCGCCGAGGCCAAAAAGAAAGCCGTGAAGGAGTCTTCTATCCGATCTGTGCAGGAGACCGTACTCCCCATCAAGAAGCGCAAGACCCGGGAGACGGTCAGCATCGAGGTCAAGGAAGTGGTGAAGCCCCTGCTGGTGTCCACCCTCGGTGAGAAGAGCGGGAAAGGACTGAAGACCTGTAAGAGCCCTGGGCGGAAAAGCAAGGAGAGCAGCCCCAAGGGGCGCAGCAGCAGCGCCTCCTCACCCCCCAAGAAGGAGCACCACCACCATCACCACCACTCAGAGTCCCCAAAGGCCCCCGTGCCACTGCTCCCACCCCTGCCCCCACCTCCACCTGAGCCCGAGAGCTCCGAGGACCCCACCAGCCCCCCTGAGCCCCAGGACTTGAGCAGCAGCGTCTGCAAAGAGGAGAAGATGCCCAGAGGAGGCTCACTGGAGAGCGACGGCTGCCCCAAGGAGCCAGCTAAGACTCAGCCCGCGGTTGCCACCGCCGCCACGGCCGCAGAAAAGTACAAACACCGAGGGGAGGGAGAGCGCAAAGACATTGTTTCATCCTCCATGCCAAGGCCAAACAGAGAGGAGCCTGTGCACAGCCGGACGCCCGTGACCGAGAGAGTTAGCGAGCAGAAGCTGATCTCAGAGGAGGACCTGTGACGACCATGGCTGTAGACTGTTACTCGAGATACATACTTCTTTACATTCCAATACATACTTCTTTACATTCCAATACATACTTCTTTACATTCCACCATGGACTAGTACAAACACCATTGTCACACTCCAACAAACACCATTGTCACACTCCAACAAACACCATTGTCACACTCCAGCGGCCGCTTCGATCCGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGCCTAGGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACAAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAG >SEQ ID NO: 11; MeCP2 in vivo construct nucleic acid sequence (scAAV-Mec229-hMeCP2-miR132(2x) miR122-1(3x) vector genome)CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGTAGCCATGCTCTAGGAAGATCAATTCGGTACAATTCACGCGTCGACAATTGAGGGCGTCACCGCTAAGGCTCCGCCCCAGCCTGGGCTCCACAACCAATGAAGGGTAATCTCGACAAAGAGCAAGGGGTGGGGCGCGGGCGCGCAGGTGCAGCAGCACACAGGCTGGTCGGGAGGGCGGGGCGCGACGTCTGCCGTGCGGGGTCCCGGCATCGGTTGCGCGCGCGCTCCCTCCTCTCGGAGAGAGGGCTGTGGTAAAACCCGTCCGGAAAATGGCTGCAGCCGCTGCCCCAGCGCCGAGCGGCGGAGGTGGCGGTGGCGAGGAGGAGAGACTGGAAGAAAAGTCAGAAGACCAGGACCTCCAGGGCCTCAAGGACAAACCCCTCAAGTTTAAAAAGGTGAAGAAAGATAAGAAAGAAGAGAAAGAGGGCAAGCATGAGCCCGTGCAGCCATCAGCCCACCACTCTGCTGAGCCCGCAGAGGCAGGCAAAGCAGAGACATCAGAAGGGTCAGGCTCCGCCCCGGCTGTGCCGGAAGCTTCTGCCTCCCCCAAACAGCGGCGCTCCATCATCCGTGACCGGGGACCCATGTATGATGACCCCACCCTGCCTGAAGGCTGGACACGGAAGGTTAAGCAAAGGAAATCTGGACGCTCTGCTGGGAAGTATGATGTGTATTTGATCAATCCCCAGGGAAAAGCCTTTCGCTCTAAAGTGGAGTTGATTGCGTACTTCGAAAAGGTAGGCGACACATCCCTGGACCCTAATGATTTTGACTTCACGGTAACTGGGAGAGGGAGCCCCTCCCGGCGAGAGCAGAAACCACCTAAGAAGCCCAAATCTCCCAAAGCTCCAGGAACTGGCAGAGGTCGGGGACGCCCCAAAGGGAGCGGCACCACGAGACCCAAGGCAGCTACGTCAGAGGGTGTGCAGGTGAAAAGGGTCCTGGAGAAAAGTCCTGGGAAGCTCCTTGTCAAGATGCCTTTTCAAACTTCGCCAGGGGGCAAGGCTGAGGGGGGTGGGGCCACCACATCCACCCAGGTCATGGTGATCAAACGCCCCGGCAGGAAGCGAAAAGCTGAGGCAGACCCTCAGGCCATTCCCAAGAAACGGGGTCGAAAGCCGGGGAGTGTGGTGGCAGCCGCTGCCGCCGAGGCCAAAAAGAAAGCCGTGAAGGAGTCTTCTATCCGATCTGTGCAGGAGACCGTACTCCCCATCAAGAAGCGCAAGACCCGGGAGACGGTCAGCATCGAGGTCAAGGAAGTGGTGAAGCCCCTGCTGGTGTCCACCCTCGGTGAGAAGAGCGGGAAAGGACTGAAGACCTGTAAGAGCCCTGGGCGGAAAAGCAAGGAGAGCAGCCCCAAGGGGCGCAGCAGCAGCGCCTCCTCACCCCCCAAGAAGGAGCACCACCACCATCACCACCACTCAGAGTCCCCAAAGGCCCCCGTGCCACTGCTCCCACCCCTGCCCCCACCTCCACCTGAGCCCGAGAGCTCCGAGGACCCCACCAGCCCCCCTGAGCCCCAGGACTTGAGCAGCAGCGTCTGCAAAGAGGAGAAGATGCCCAGAGGAGGCTCACTGGAGAGCGACGGCTGCCCCAAGGAGCCAGCTAAGACTCAGCCCGCGGTTGCCACCCCCGCCACGGCCGCAGAAAAGTACAAACACCGAGGGGAGGGAGAGCGCAAAGACATTGTTTCATCCTCCATGCCAAGGCCAAACAGAGAGGAGCCTGTGGACAGCCGGACGCCCGTGACCGAGAGAGTTAGCGAGCAGAAGCTGATCTCAGAGGAGGACCTGTGACGACCATGGCTGTAGACTGTTACGACCATGGCTGTAGACTGTTACTCGAGATACATACTTGTTTACATTCCAATACATACTTCTTTACATTCCAATACATACTTCTTTACATTCCACCATGGACTAGTACAAACACCATTGTCACACTCCAACAAACACCATTGTCACACTCCAACAAACACCATTGTCACACTCCAGCGGCCGCTTCGATCCGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGCCTAGGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACAAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAG >SEQ ID NO: 12; MeCP2 in vivo construct nucleic acid sequence (scAAV-Mec229-hMeCP2-miR132(3x) miR 122-1(3x) vector genome)CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGTAGCCATGCTCTAGGAAGATCAATTCGGTACAATTCACGCGTCGACAATTGAGGGCGTCACCGCTAAGGCTCCGCCCCAGCCTGGGCTCCACAACCAATGAAGGGTAATCTCGACAAAGAGCAAGGGGTGGGGCGCGGGCGCGCAGGTGCAGCAGCACACAGGCTGGTCGGGAGGGCGGGGCGCGACGTCTGCCGTGCGGGGTCCCGGCATCGGTTGCGCGCGCGCTCCCTCCTCTCGGAGAGAGGGCTGTGGTAAAACCCGTCCGGAAAATGGCTGCAGCCGCTGCCGCAGCGCCGAGCGGCGGAGGTGGCGGTGGCGAGGAGGAGAGACTGGAAGAAAAGTCAGAAGACCAGGACCTCCAGGGCCTCAAGGACAAACCCCTCAAGTTTAAAAAGGTGAAGAAAGATAAGAAAGAAGAGAAAGAGGGCAAGCATGAGCCCGTGCAGCCATCAGCCCACCACTCTGCTGAGCCCGCAGAGGCAGGCAAAGCAGAGACATCAGAAGGGTCAGGCTCCGCCCCGGCTGTGCCGGAAGCTTCTGCCTCCCCCAAACAGCGGCGCTCCATCATCCGTGACCGGGGACCCATGTATGATGACCCCACCCTGCCTGAAGGCTGGACACGGAAGCTTAAGCAAAGGAAATCTGGACGCTCTGCTGGGAAGTATGATGTGTATTTGATCAATCCCCAGGGAAAAGCCTTTCGCTCTAAAGTGGAGTTGATTGCGTACTTCGAAAAGGTAGGCGACACATCCCTGGACCCTAATGATTTTGACTTCACGGTAACTGGGAGAGGGAGCCCCTCCCGGCGAGAGCAGAAACCACCTAAGAAGCCCAAATCTCCCAAAGCTCCAGGAACTGGCAGAGGTCGGGGACGCCCCAAAGGGAGCGGCACCACGAGACCCAAGGCAGCTACGTCAGAGGGTGTGCAGGTGAAAAGGGTCCTGGAGAAAAGTCCTGGGAAGCTCCTTGTCAAGATGCCTTTTCAAACTTCGCCAGGGGGCAAGGCTGAGGGGGGTGGGGCCACCACATCCACCCAGGTCATGGTGATCAAACGCCCCGGCAGGAAGCGAAAAGCTGAGGCAGACCCTCAGGCCATTCCCAAGAAACGGGGTCGAAAGCCGGGGAGTGTGGTGGCAGCCGCTGCCGCCGAGGCCAAAAAGAAAGCCGTGAAGGAGTCTTCTATCCGATCTGTGCAGGAGACCGTACTCCCCATCAAGAAGCGCAAGACCCGGGAGACGGTCAGCATCGAGGTCAAGGAAGTGGTGAAGCCCCTGCTGGTGTCCACCCTCGGTGAGAAGAGCGGGAAAGGACTGAAGACCTGTAAGAGCCCTGGGCGGAAAAGCAAGGAGAGCAGCCCCAAGGGGCGCAGCAGCAGCGCCTCCTCACCCCCCAAGAAGGAGCACCACCACCATCACCACCACTCAGAGTCCCCAAAGGCCCCCGTGCCACTGCTCCCACCCCTGCCCCCACCTCCACCTGAGCCCGAGAGCTCCGAGGACCCCACCAGCCCCCCTGAGCCCCAGGACTTGAGCAGCAGCGTCTGCAAAGAGGAGAAGATGCCCAGAGGAGGCTCACTGGAGAGCGACGGCTGCCCCAAGGAGCCAGCTAAGACTCAGCCCGGGGTTGCCACCGCCGCCACGGCCGCAGAAAAGTACAAACACCGAGGGGAGGGAGAGCGCAAAGACATTGTTTCATCCTCCATGCCAAGGCCAAACAGAGAGGAGCCTGTGGACAGCCGGACGCCCGTGACCGAGAGAGTTAGCGAGCAGAAGCTGATCTCAGAGGAGGACCTGTGACGACCATGGCTGTAGACTGTTACGACCATGGCTGTAGACTGTTACGACCATGGCTGTAGACTGTTACTCGAGATACATACTTCTTTACATTCCAATACATACTTCTTTACATTCCAATACATACTTCTTTACATTCCACCATGGACTAGTACAAACACCATTGTCACACTCCAACAAACACCATTGTCACACTCCAACAAACACCATTGTCACACTCCAGCGGCCGCTTCGATCCGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGCCTAGGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACAAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTCCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAG >SEQ ID NO: 13; AAV2 capsid amino acid sequenceMAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDINGVYSEPRPIGTRYLTRNL >SEQ ID NO: 14; AAV9 capsid amino acid sequenceMAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL >SEQ ID NO: 15; AAV-PHP.B capsid amino acid sequenceMAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDPNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQTLAVPFKAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL

What is claimed is:
 1. A method of engineering a transgene, the methodcomprising: (a) selecting a first gene encoding a first product in acell; (b) selecting an miRNA, the expression of which is positivelyregulated by the first product in the cell; and, (c) engineering atransgene that expresses a transcript having a coding region encodingthe first product and a 3′-non-coding region comprising one or morebinding sites for the miRNA.
 2. A method of engineering a transgene, themethod comprising: (a) selecting a first gene encoding a first productin a cell; (b) selecting a second gene encoding a second product in thecell; (c) determining that expression of the second product ispositively regulated by the first product in the cell; (d) selecting anmiRNA; (e) determining that expression of the miRNA is positivelyregulated by the second product in the cell; and, (f) engineering atransgene to express in the cell a transcript having a coding regionencoding the first product and a 3′-non-coding region comprising one ormore binding sites for the miRNA.
 3. The method of claim 1 or 2, whereinthe first product is a protein.
 4. The method of claim 3, wherein theprotein is MeCP2, optionally MeCP2 isoform e1 or MeCP isoform e2.
 5. Themethod of any one of claims 2 to 4, wherein the second product is aprotein, or nucleic acid, optionally wherein the nucleic acid is anmiRNA.
 6. The method of any one of claims 1 to 5, wherein the miRNA ismiR-132.
 7. The method of any one of claims 1 to 6, wherein the methodfurther comprises engineering the 3′-non-coding region of the transcriptto comprise one or more binding sites for one or more de-targetingmiRNAs.
 8. The method of claim 7, wherein the one or more de-targetingmiRNAs inhibit expression of the transgene from liver, heart, lung,muscle, pancreas, or antigen presenting cells.
 9. The method of claim 7or 8, wherein the one or more de-targeting miRNA is miR-122, miR-1, ormiR-122 and miR-1.
 10. The method of any one of claims 1 to 9, whereinthe step of engineering the transgene comprises inserting the transgeneinto a vector.
 11. The method of claim 10, wherein the vector is acloning vector, expression vector, plasmid, or viral vector.
 12. Amethod of engineering a transgene, the method comprising: (a) selectinga first gene encoding a first product in a cell; (b) selecting an miRNA,the expression of which is positively regulated by the first product inthe cell; and, (c) engineering a transgene that expresses a transcripthaving a coding region encoding the first product and having one or morebinding sites for the miRNA.
 13. A method of engineering a transgene,the method comprising; (a) selecting a first gene encoding a firstproduct in a cell; (b) selecting a second gene encoding a second productin the cell; (c) determining that expression of the second product ispositively regulated by the first product in the cell; (d) selecting anmiRNA; (e) determining that expression of the miRNA is positivelyregulated by the second product in the cell; and, (f) engineering atransgene to express in the cell a transcript having a coding regionencoding the first product and having one or more binding sites for themiRNA.
 14. A recombinant AAV (rAAV) vector for self-regulated expressionof a protein, the rAAV vector comprising a nucleic acid engineered toexpress in a cell of a target tissue a transcript encoding the protein,wherein the transcript comprises at least one first miRNA binding sitespecific for a first miRNA, wherein expression of the first miRNA ispositively regulated by expression of the protein in the cell.
 15. Arecombinant AAV comprising a capsid harboring the rAAV vector of claim14, wherein the capsid comprises a capsid protein that facilitatesselective transduction of the cell of the target tissue.
 16. Arecombinant nucleic acid encoding a transcript having i) a coding regionencoding a protein and ii) two or more miRNA binding sites, wherein thetwo or more miRNA binding sites comprise: (a) at least one first miRNAbinding site specific for a first miRNA that is positively regulated byexpression of the protein in a cell of a target tissue; and (b) at leastone second miRNA binding site specific for a second miRNA that isexpressed, independent of expression of the protein, in cells of anon-target tissue.
 17. A recombinant nucleic acid encoding a transcripthaving a coding region encoding human MeCP2 protein or a functionalfragment thereof and a 3′-non-coding region comprising two or more miRNAbinding sites, wherein the two or more miRNA binding sites comprise: (a)at least one miRNA binding site specific for an miRNA that negativelyregulates expression of the transcript; and (b) at least one miRNAbinding site specific for an miRNA that inhibits expression of thetranscript in a cell of a non-target tissue.
 18. The recombinant nucleicacid of claim 17, wherein the coding region encodes MeCP2 isoform e1.19. The recombinant nucleic acid of claim 17 or 18, wherein the humanMeCP2 comprises the sequence set forth in SEQ ID NO:
 1. 20. Therecombinant nucleic acid of any one of claims 17 to 19, wherein the atleast one miRNA binding site specific for an miRNA that negativelyregulates expression of the transcript comprises a miR-132 binding site,optionally wherein the at least one miRNA binding site is two or threemiR-132 binding sites.
 21. The recombinant nucleic acid of any one ofclaims 17 to 20, wherein the at least one miRNA binding site specificfor an miRNA that inhibits expression of the transcript in a non-targettissue comprises a miR-1 binding site, mir-122 binding site, or miR-1and miR-122 binding site.
 22. The recombinant nucleic acid of any one ofclaims 17 to 21, wherein each of the one or more miRNA binding sites islocated between the last codon of the coding region and the poly-A tailof the transcript.
 23. The recombinant nucleic acid of any one of claims17 to 22, further comprising a promoter, optionally a mouse MeCP2promoter.
 24. The recombinant nucleic acid of claim 23, wherein themouse MeCP2 promoter comprises the sequence set forth in SEQ ID NO: 3.25. The recombinant nucleic acid of any one of claims 17 to 24, whereinthe recombinant nucleic acid is located on a plasmid.
 26. A viral vectorcomprising the recombinant nucleic acid of any one of claims 17 to 24,optionally wherein the viral vector is an adeno-associated virus (AAV)vector, an adenovirus vector, a lentiviral vector, a herpesvirus vector,or a baculovirus vector.
 27. A recombinant nucleic acid encoding atranscript having (a) a coding region encoding human MeCP2 or afunctional fragment thereof; and, (b) a 3′-non-coding region comprisingone or more miRNA binding sites, wherein transcript is flanked byadeno-associated virus (AAV) inverted terminal repeats (ITRs).
 28. Therecombinant nucleic acid of claim 27, wherein the coding region encodesMeCP2 isoform e1.
 29. The recombinant nucleic acid of claim 27 or 28,wherein the human MeCP2 comprises the sequence set forth in SEQ IDNO:
 1. 30. The recombinant nucleic acid of any one of claims 27 to 29,wherein the one or more miRNA binding sites are miR-1, miR-122, ormiR-132 binding sites, or any combination thereof.
 31. The recombinantnucleic acid of claim 30, wherein the transcript comprises a miR-1binding site, a mir-122 binding site, and at least one miR-132 bindingsite.
 32. The recombinant nucleic acid of claim 31, wherein the at leastone miR-132 binding site is two or three mir-132 binding sites.
 33. Therecombinant nucleic acid of any one of claims 27 to 32, wherein the oneor more miRNA binding sites are located between the last codon of thecoding region and the poly-A tail of the transcript.
 34. The recombinantnucleic acid of any one of claims 27 to 33, further comprising apromoter, optionally a mouse MeCP2 promoter.
 35. The recombinant nucleicacid of claim 34, wherein the mouse MeCP2 promoter comprises thesequence set forth in SEQ ID NO:
 3. 36. The recombinant nucleic acid ofany one of claims 27 to 35, wherein the ITRs are AAV2 ITRs.
 37. Arecombinant adeno-associated virus (rAAV) comprising: a capsid harboringthe recombinant nucleic acid of any one of claims 17 to 24, or 27 to 36.38. The rAAV of claim 37, wherein the nucleic acid comprises at leastone ITR selected from an AAV2, AAV3, AAV4, AAV5, or AAV6 ITR.
 39. TherAAV of claim 37 or 38, wherein the capsid comprises a capsid proteinthat facilitates passage of the rAAV across the blood-brain barrier. 40.The rAAV of claim 39, wherein the capsid protein has a serotype selectedfrom the group consisting of AAV-PHP.B, AAV1, AAV2, AAV2i8, AAV2.5,AAV5, AAV6, AAV8, AAVrh8, AAV9, AAVrh10, AAV-B1, AAV9.45A-String (e.g.,AAV9.45-AS), AAV9.45Angiopep, AAV9.47-Angiopep, AAV9.47-AS, AAV-CAM130,and AAV9HR.
 41. The rAAV of any one of claims 37 to 40, wherein thecapsid protein comprises or consists of a sequence set forth in SEQ IDNO: 14 or 15 (AAV-PHP.B, AAV9).
 42. A composition comprising therecombinant nucleic acid of any one of claims 17 to 24, or 27 to 36, orthe rAAV of any one of claims 37 to 41, and a pharmaceuticallyacceptable excipient.
 43. The composition of claim 42, wherein thecomposition is formulated for injection, optionally wherein theinjection is systemic injection (e.g., intravenous injection) orintrathecal injection.
 44. A method of treating Rett syndrome in asubject, the method comprising, administering to a subject having orsuspected of having Rett syndrome an effective amount of: (a) therecombinant nucleic acid of any one of claims 17 to 24, or 27 to 36; (b)the rAAV of any one of claims 37 to 41; or, (c) the composition of claim42 or
 43. 45. The method of claim 44, wherein the subject is a humansubject, optionally wherein the subject is less than one year old. 46.The method of claim 44 or 45, wherein the subject is characterized by amutation in at least one copy of the MeCP2 gene, optionally wherein themutation is a loss of function mutation.
 47. The method of any one ofclaims 44 to 46, wherein the administration is injection, optionallysystemic injection (e.g., intravenous injection) or intrathecalinjection.
 48. The method of any one of claims 44 to 47, wherein theadministration results in the effective amount of (a), (b), or (c)crossing the blood-brain barrier of the subject.
 49. The method of anyone of claims 44 to 48, wherein the administration results in anon-toxic level of MeCP2 expression in the brain of the subject.